From delivani at mpi-cbg.de  Mon Jan  9 13:25:55 2012
From: delivani at mpi-cbg.de (Petrina Delivani)
Date: Mon, 9 Jan 2012 14:25:55 +0100
Subject: [Pombelist] TetR plasmid
Message-ID: <04CAB867-EE57-429C-93C9-DC9687EAB221@mpi-cbg.de>

Hello pombe fans!

I would like to label pombe chromosomes with the tetO system. 
Does anybody have a plasmid containing the tetR-NLS-floursescence marker sequence? And would be happy to spare me some?

Thanks, Petrina.



Petrina Delivani, PhD
Tolic-Norrelykke-Lab
MPI-CBG, Dresden
Pfotenhauerstr. 108
01307 Dresden

delivani at mpi-cbg.de
Tel: 0049-351-210-2479
Fax: 0049-351-210-2020
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120109/8b0ac106/attachment.htm>

From forsburg at usc.edu  Tue Jan 10 01:07:31 2012
From: forsburg at usc.edu (SLForsburg)
Date: Mon, 9 Jan 2012 17:07:31 -0800
Subject: [Pombelist] Looking for inert genomic locus
Message-ID: <ACE7D910-CBCD-4F9C-A095-4027E0AA8954@usc.edu>

We want to use an inert sequence for a chromatin immunoprecipitation experiment.  It must be in the euchromatin, distant from replication origins,  and not transcribed (no genes, no ncRNA).  It's probably the most boring part of the genome.   It would be an added bonus if anyone has designed ChIP primers for it.  

Any suggestions?  I will summarize and post.

Thanks!

Susan F.

PS:  the pombe shop at cafepress has a new design:  the pombe international tour tee-shirt!

http://www.cafepress.com/+pombe_international_tour_white,606010033


 
{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 




-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120109/aecb5908/attachment.htm>

From burcinaltun at gmail.com  Wed Jan 11 14:46:16 2012
From: burcinaltun at gmail.com (burcin altun)
Date: Wed, 11 Jan 2012 15:46:16 +0100
Subject: [Pombelist] (no subject)
Message-ID: <CA+5fPiOZX6jzs0K0tNw6VgK7cqW1gaG29M8kxyCDgPZXH7DFbg@mail.gmail.com>

Dear Pombe People,
I would like to inform about YAC clonning, As i know people use yac just
for big size inserts, but i m planning to use it just for 3genes and
totally my insert will be 1000nukleotid. Is there anyone who knows anything
about clonning small size inside YAC? Is it stable?
Thanks in advance
-- 

Burcin ALTUN
PhD Student
Yeast Molecular Genetics Group
International Centre for Genetic Engineering and Biotechnology (ICGEB)
Padriciano 99, 34012 Trieste, Italy
E-mail:   altun at icgeb.org <passos at icgeb.org>
Phone:  +39-340-8613728



*
*
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120111/7d22dded/attachment.htm>

From burcinaltun at gmail.com  Wed Jan 11 16:23:43 2012
From: burcinaltun at gmail.com (burcin altun)
Date: Wed, 11 Jan 2012 17:23:43 +0100
Subject: [Pombelist] YAC clonning
Message-ID: <CA+5fPiMQXupyctUEGHdNG46ufsCaUR+QRAXV+DWe8LNe=nBTzA@mail.gmail.com>

Dear Pombe People,
I would like to inform about YAC clonning, As i know people use yac just
for big size inserts, but i m planning to use it just for 3genes and
totally my insert will be 1000nukleotid. Is there anyone who knows anything
about clonning small size inside YAC? Is it stable?
Thanks in advance

-- 

Burcin ALTUN
PhD Student
Yeast Molecular Genetics Group
International Centre for Genetic Engineering and Biotechnology (ICGEB)
Padriciano 99, 34012 Trieste, Italy
E-mail: altun at icgeb.org
Phone:  +39-340-8613728



*
*
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120111/70d36f3a/attachment.htm>

From sponberg1 at gmail.com  Fri Jan 13 13:39:40 2012
From: sponberg1 at gmail.com (=?ISO-8859-1?Q?Bj=F8rn_sponberg?=)
Date: Fri, 13 Jan 2012 14:39:40 +0100
Subject: [Pombelist] List of all Convergent Gene pairs in S. Pombe
Message-ID: <CAHgjbtRAL=sokSQPgM7_1U4=QztS+XUvZHJNtQ2C-nwo-FizLA@mail.gmail.com>

Hi all,
I have read much about Convergent Gene pairs in S. Pombe. They should make
up about ~30% of all protein coding genes. Does anyone know of a list that
contain CG exclusively?
Thanks!


Cheers
Bj?rn

Bj?rn Sponberg M.Sc.
Department of Biochemistry and Biophysics
Stockholm University
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/d5ab8c64/attachment.htm>

From punitprasad at gmail.com  Fri Jan 13 14:03:21 2012
From: punitprasad at gmail.com (Punit Prasad)
Date: Fri, 13 Jan 2012 15:03:21 +0100
Subject: [Pombelist] Inducible system
Message-ID: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>

Hello,
I am looking for non-leaky inducible promoter system in pombe. Can I get
some suggestions from the experts.
Thanks in advance
Punit

-- 
*Punit Prasad*
Post Doctoral Fellow
Dept of Bioscience and Nutrition
Karolinska Institute
Stockholm
Sweden
http://www.bionut.ki.se/groups/kek/
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/66ae8639/attachment.htm>

From hoffmacs at bc.edu  Fri Jan 13 14:11:30 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Fri, 13 Jan 2012 09:11:30 -0500
Subject: [Pombelist] Inducible system
In-Reply-To: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
References: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
Message-ID: <DA8C3520-A26B-4C61-986C-5996A5372540@bc.edu>

urg1 may be the best system, but you have to put your gene into the urg1 locus.

http://www.ncbi.nlm.nih.gov/pubmed/21664261


fbp1 is also very good in liquid culture, but since the promoter turns on in stationary phase, it is will come on in cells in a colony on a plate.  It will also be tighter in an integrated single copy than on a multicopy plasmid, but it does not have to be at the fbp1 locus. 

http://www.ncbi.nlm.nih.gov/pubmed/2558974

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"



On Jan 13, 2012, at 9:03 AM, Punit Prasad wrote:

> Hello,
> I am looking for non-leaky inducible promoter system in pombe. Can I get some suggestions from the experts. 
> Thanks in advance
> Punit
> 
> -- 
> Punit Prasad
> Post Doctoral Fellow
> Dept of Bioscience and Nutrition
> Karolinska Institute
> Stockholm
> Sweden
> http://www.bionut.ki.se/groups/kek/
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist



-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/fedb0861/attachment.htm>

From a.m.carr at sussex.ac.uk  Fri Jan 13 16:09:23 2012
From: a.m.carr at sussex.ac.uk (Antony Carr)
Date: Fri, 13 Jan 2012 16:09:23 +0000
Subject: [Pombelist] Inducible system
In-Reply-To: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
References: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
Message-ID: <C5AD4BE27ED4F042BF57EA254C30BC2228CF0BD8@EX-SHA-MBX1.ad.susx.ac.uk>

Charlie is correct, you could consider urg1. We have a number of stains and plasmids to help described in the link he sent.

Also, we have adapted it to reduce expression levels (unpublished) and you are welcome to try that as well.

Tony

Tony Carr
Director, GDSC
University of Sussex
01273 678122

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Punit Prasad
Sent: 13 January 2012 14:03
To: pombelist at sanger.ac.uk
Subject: [Pombelist] Inducible system

Hello,
I am looking for non-leaky inducible promoter system in pombe. Can I get some suggestions from the experts.
Thanks in advance
Punit

--
Punit Prasad
Post Doctoral Fellow
Dept of Bioscience and Nutrition
Karolinska Institute
Stockholm
Sweden
http://www.bionut.ki.se/groups/kek/
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/83c18655/attachment.htm>

From MEC at stowers.org  Fri Jan 13 18:11:48 2012
From: MEC at stowers.org (Cook, Malcolm)
Date: Fri, 13 Jan 2012 12:11:48 -0600
Subject: [Pombelist] List of all Convergent Gene pairs in S. Pombe
In-Reply-To: <CAHgjbtRAL=sokSQPgM7_1U4=QztS+XUvZHJNtQ2C-nwo-FizLA@mail.gmail.com>
References: <CAHgjbtRAL=sokSQPgM7_1U4=QztS+XUvZHJNtQ2C-nwo-FizLA@mail.gmail.com>
Message-ID: <2C40E43D1F7A56408C4463FD245DDDF995638CD7@EXCHMB-02.stowers-institute.org>

Bjorn,

I have been building out genomic resources in S. Pombe in support of a research program, and have used this as an example to demo to a collaborator the value of using R/Bioconductor for posing such questions.

I hope you find informative the attached BED formatted file listing 2146 convergent pairs.  You should be able to open it in a text editor, excel, or upload it for display it at pombase or ensembl (http://fungi.ensembl.org/Schizosaccharomyces_pombe/)

Cheers,

~Malcolm

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Bj?rn sponberg
Sent: Friday, January 13, 2012 7:40 AM
To: pombelist at sanger.ac.uk
Subject: [Pombelist] List of all Convergent Gene pairs in S. Pombe

Hi all,
I have read much about Convergent Gene pairs in S. Pombe. They should make up about ~30% of all protein coding genes. Does anyone know of a list that contain CG exclusively?
Thanks!


Cheers
Bj?rn

Bj?rn Sponberg M.Sc.
Department of Biochemistry and Biophysics
Stockholm University
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/190f2667/attachment-0001.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: pombeConvergentTx.bed
Size: 168780 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/190f2667/pombeConvergentTx-0001.bed>

From bassem.alsady at gmail.com  Fri Jan 13 18:32:54 2012
From: bassem.alsady at gmail.com (Bassem Al-Sady)
Date: Fri, 13 Jan 2012 10:32:54 -0800
Subject: [Pombelist] pombe FPs
Message-ID: <4F1078D6.8030104@ucsf.edu>

Hi,

I am looking for fluorescent proteins that behave well in pombe for flow 
cytometry, esp. if anyone has tested the newer generation of orange and 
red proteins.

thank you,

Bassem

-- 
Bassem Al-Sady, Ph.D.
Postdoctoral Fellow
Department of Biochemistry and Biophysics
University of California, San Francisco
Genentech Hall, MC 2240
600 16th St, San Francisco CA 94158
1-415-514-4302 (lab)



From jag at imtech.res.in  Sat Jan 14 09:53:22 2012
From: jag at imtech.res.in (jag at imtech.res.in)
Date: Sat, 14 Jan 2012 15:23:22 +0530 (IST)
Subject: [Pombelist] ade6 transcription start site
Message-ID: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>

Hi:

We are intersted in manipulating ade6 gene but would like to be careful about
disrupting the transcription start and termination sites. Any information about
the location of these sites would be welcome.
Thanks
Jag

______________________________________________________________________
?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
?????? 39 ?, ???????? / Sector 39-A, Chandigarh
??? ???/PIN CODE :160036
??????/EPABX :0172 6665 201-202


From francis.stewart at biotec.tu-dresden.de  Sat Jan 14 20:08:18 2012
From: francis.stewart at biotec.tu-dresden.de (Francis Stewart)
Date: Sat, 14 Jan 2012 21:08:18 +0100
Subject: [Pombelist] Inducible system
In-Reply-To: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
References: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
Message-ID: <68FB954E-F704-4F6B-BBB2-17017E47F218@biotec.tu-dresden.de>

Punit

you might like to look at the data in this paper, in particular Figure 2.

regards,

Francis



On 13 Jan 2012, at 15:03, Punit Prasad wrote:

> Hello,
> I am looking for non-leaky inducible promoter system in pombe. Can I get some suggestions from the experts. 
> Thanks in advance
> Punit
> 
> -- 
> Punit Prasad
> Post Doctoral Fellow
> Dept of Bioscience and Nutrition
> Karolinska Institute
> Stockholm
> Sweden
> http://www.bionut.ki.se/groups/kek/
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist


Prof. A. Francis Stewart,
Genomics, Technische Universitaet Dresden
BioInnovationZentrum
Tatzberg 47
01307 Dresden
tel +49-351-46340129 or 30
fax+49-351-46340143
http://www.biotec.tu-dresden.de/research-faculty/stewart/group-page/

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120114/a12ddce7/attachment-0002.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: Erler Yeast.pdf
Size: 285565 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120114/a12ddce7/ErlerYeast-0001.pdf>
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120114/a12ddce7/attachment-0003.htm>

From val at sanger.ac.uk  Mon Jan 16 10:18:58 2012
From: val at sanger.ac.uk (Val Wood)
Date: Mon, 16 Jan 2012 10:18:58 +0000
Subject: [Pombelist] ade6 transcription start site
In-Reply-To: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
Message-ID: <4F13F992.2010408@sanger.ac.uk>


Hello Jag,

The UTRS of ade6 appear to be short, and are represented in this view,
http://www.pombase.org/spombe/result/SPCC1322.13 as short "open boxes"
as derived from Rhind et al  transcriptome data PMID:21511999

// B?hler la transcriptome viewer for ade6
http://www.bahlerlab.info/cgi-bin/pombetv/pombetv?condition=13&genename=SPCC1322.13&action=display
and EST data indicates a slightly shorter transcript beginning at 
1316291 and ending at  1318035

I have updated the "consensus UTRs" to be based on these coordinates 
supported by ESTs FY124947 and FY087729

The information  used to derive the consensus UTRs will be displayed 
under the "Transcipt" section of the gene page shortly.

Val




On 14/01/2012 09:53, jag at imtech.res.in wrote:
> Hi:
>
> We are intersted in manipulating ade6 gene but would like to be careful about
> disrupting the transcription start and termination sites. Any information about
> the location of these sites would be welcome.
> Thanks
> Jag
>
> ______________________________________________________________________
> ?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
> Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
> ?????? 39 ?, ???????? / Sector 39-A, Chandigarh
> ??? ???/PIN CODE :160036
> ??????/EPABX :0172 6665 201-202
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120116/93033f1a/attachment.htm>

From Nick.Rhind at umassmed.edu  Tue Jan 17 15:17:27 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Tue, 17 Jan 2012 15:17:27 +0000
Subject: [Pombelist] ade6 transcription start site
In-Reply-To: <4F13F992.2010408@sanger.ac.uk>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
	<4F13F992.2010408@sanger.ac.uk>
Message-ID: <544C3935-E0C9-478E-9C83-D6594F82FEA6@umassmed.edu>

Hi Jag,

Another resource you might want to investigate is an annotation of UTRs in Table S2 of Lantermann et al. <http://www.ncbi.nlm.nih.gov/pubmed/20118936>, based on the HybMap data from Dutrow et al. <http://www.ncbi.nlm.nih.gov/pubmed/18641648>.

Nick


On 16 Jan, 12, at 5:18 AM, Val Wood wrote:

> 
> Hello Jag,
> 
> The UTRS of ade6 appear to be short, and are represented in this view,
> http://www.pombase.org/spombe/result/SPCC1322.13 as short "open boxes"
> as derived from Rhind et al  transcriptome data PMID:21511999  
> 
> B?hler la transcriptome viewer for ade6
> http://www.bahlerlab.info/cgi-bin/pombetv/pombetv?condition=13&genename=SPCC1322.13&action=display
> and EST data indicates a slightly shorter transcript beginning at 1316291 and ending at  1318035
> 
> I have updated the "consensus UTRs" to be based on these coordinates supported by ESTs FY124947 and FY087729
> 
> The information  used to derive the consensus UTRs will be displayed under the "Transcipt" section of the gene page shortly.
> 
> Val
> 
> 
> 
> 
> On 14/01/2012 09:53, jag at imtech.res.in wrote:
>> Hi:
>> 
>> We are intersted in manipulating ade6 gene but would like to be careful about
>> disrupting the transcription start and termination sites. Any information about
>> the location of these sites would be welcome.
>> Thanks
>> Jag
>> 
>> ______________________________________________________________________
>> ?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
>> Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
>> ?????? 39 ?, ???????? / Sector 39-A, Chandigarh
>> ??? ???/PIN CODE :160036
>> ??????/EPABX :0172 6665 201-202
>> 
>> _______________________________________________
>> Pombelist mailing list
>> 
>> Pombelist at sanger.ac.uk
>> http://lists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From adele.marston at ed.ac.uk  Wed Jan 18 11:32:02 2012
From: adele.marston at ed.ac.uk (Adele Marston)
Date: Wed, 18 Jan 2012 11:32:02 +0000
Subject: [Pombelist] British Yeast Group Meeting - REGISTRATION IS OPEN
Message-ID: <304023AF-2CBB-486F-B22A-3F10C8DEDD37@ed.ac.uk>

Dear Pombe people,

REGISTRATION IS NOW OPEN for the 2012 BYG meeting in Edinburgh (21st-23rd March).

Costs are ?260 all inclusive for single occupancy room.
If you don't need accommodation, it's ?170 (all meals etc. are provided).
There is a discount for genetics society members.

Please register NOW at (<http://www.britishyeastgroup.org/>)
Abstracts should be sent to: byg2012 at britishyeastgroup.org (instructions are on the website) BEFORE 17 February.
The majority of the talks will be selected from the abstracts. If you do not wish to be considered for a talk, please let us know when you send your abstract.

We also have an exciting programme of invited speakers:

John Kilmartin (Keynote Lecture)
Jurg Bahler (Global gene expression)
Tony Carr (DNA damage)
Anne Donaldson (DNA replication)
Iain Hagan (Mitosis)
Jon Houseley (Non-coding RNA)
Ed Louis (Telomeres, genome evolution)
Carol Munro (Fungal pathogens)
Markus Ralser (Metabolic networks)
Ken Sawin (Microtubules)
Martin Singleton (Kinetochore structures)
Tomo Tanaka (Chromosome Segregation)

The meeting will take place in the University's conference centre. The conference space is large and open with plenty of space for posters and mingling over drinks. All accommodation is single occupancy (double room) with ensuite facilities. The venue is in a beautiful part of Edinburgh, close to Holyrood park and walking distance from the centre. 

As well as the conference dinner, we will have some traditional Scottish entertainment and have secured sufficient sponsorship to ensure a very entertaining meeting both scientifically and socially.

I look forward to seeing you in Edinburgh in March!

Adele

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120118/85b12c02/attachment.htm>
-------------- Attachments --------------
An embedded and charset-unspecified text was scrubbed...
Name: not available
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120118/85b12c02/attachment.asc>

From luis.rokeach at umontreal.ca  Thu Jan 19 18:52:21 2012
From: luis.rokeach at umontreal.ca (Rokeach Luis)
Date: Thu, 19 Jan 2012 18:52:21 -0000
Subject: [Pombelist] postdoc position
Message-ID: <C83F51F1.D388%luis.rokeach@umontreal.ca>

Dear Colleagues,
 
I would greatly appreciate your posting of this ad for a postdoc position in
my lab.
 
 


With best regards,
 

Luis
 
Luis A. Rokeach, PhD
Professor
Department of Biochemistry
Universit? de Montr?al
C.P. 6128, succ. Centre-ville
Phone 1(514) 343-6324; Fax 1(514) 343-2210
luis.rokeach at umontreal.ca <mailto:luis.rokeach at umontreal.ca>
http://mapageweb.umontreal.ca/rokeach
 
 
 


-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120119/dfb8b105/attachment.htm>

From Pernilla.Bjerling at imbim.uu.se  Fri Jan 20 14:41:45 2012
From: Pernilla.Bjerling at imbim.uu.se (Pernilla Bjerling)
Date: Fri, 20 Jan 2012 15:41:45 +0100
Subject: [Pombelist] post-doc
Message-ID: <FE9E83FC-D326-4597-A2FB-CA87FB8D3CE5@imbim.uu.se>

To retrieve the following attachment, use the link provided
Name: Postdoc_epigenetics.doc
Size: 56320 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120120/a5d2b187/Postdoc_epigenetics-0001.doc>
-------------- Attachments --------------


Dear All

I am hiring a post-doc (see the ad) AND a PhD student so please post  
and/or forward this to suitable candidates.

The best! / Pernilla

Pernilla Bjerling, PhD, Researcher
University of Uppsala
Dept. of Medical Biochemistry and Microbiology (IMBIM)
Box 582; SE-751 23 Uppsala; Sweden
Tel.: +46 18 471 6652 or +46 18 471 4243
Fax.: +46 18 471 4673
e-mail: pernilla.bjerling at imbim.uu.se
web page: http://www.imbim.uu.se/forskning/bjerlingresearch.html






From levinpombe at gmail.com  Tue Jan 24 18:56:47 2012
From: levinpombe at gmail.com (Levin Henry)
Date: Tue, 24 Jan 2012 13:56:47 -0500
Subject: [Pombelist] protocol for transforming 96 pombe strains at once
In-Reply-To: <4F13F992.2010408@sanger.ac.uk>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
	<4F13F992.2010408@sanger.ac.uk>
Message-ID: <0B299C4F-0880-43F4-AA4D-B3F5A683FA48@mail.nih.gov>

Hello All,
	We are trying to transform one plasmid into the Bioneer collection of 3000 haploid strains.  We have tried many of the protocols used to transform the S. cerevisiae deletion set and they are not working with sufficient efficiency.  I would appreciate hearing from anyone who has such a protocol. Thanks for your help.

Henry Levin
NIH, bld 18T, room 106



From drojass at med.uchile.cl  Wed Jan 25 20:23:46 2012
From: drojass at med.uchile.cl (Diego Rojas Soto)
Date: Wed, 25 Jan 2012 17:23:46 -0300 (CLST)
Subject: [Pombelist] pombe expression vector and strain
Message-ID: <65270.172.16.77.175.1327523026.squirrel@correo.med.uchile.cl>

Hello:

I would like express a transcription factor in pombe cells to study the
effect in ribosomal protein expression but the vector has a ura+ marker so
I need the pombe strain MP6-10B. Where can I get it?

Best regards


Diego Rojas Soto
University of Chile
Faculty of Medicine


From pei-yun.wu at univ-rennes1.fr  Tue Jan 31 12:20:47 2012
From: pei-yun.wu at univ-rennes1.fr (Jenny Wu)
Date: Tue, 31 Jan 2012 13:20:47 +0100
Subject: [Pombelist] Post-doctoral position
Message-ID: <E79194B3-0650-460C-97F2-698A0B8B581B@univ-rennes1.fr>

Hi everyone,

A post-doctoral position is available in my laboratory, the Genome Duplication and Maintenance group, at the Institute of Genetics and Development of Rennes (IGDR) in Brittany, France. Our team uses fission yeast to investigate the control of DNA replication and the role of the multiple layers regulating DNA synthesis in the maintenance of genome integrity. We are seeking candidates to conduct a project that will focus on the fundamental parameters that determine the organization and selection of replication origins. This position is supported by the French Foundation for Medical Research.

For further information regarding the position and the institute, please see the attached PDF file as well as the following website: 

http://igdr.univ-rennes1.fr/english/


Please contact me if you need any additional information, and thank you for your time.



Sincerely,

Jenny Wu

**************************************************************************
Laboratory of Genome Duplication and Maintenance
Institute of Genetics and Development of Rennes, CNRS UMR 6290
2 av du Professeur L?on Bernard
Rennes, France 35043
email: pei-yun.wu at univ-rennes1.fr
phone: +33 2 23 23 44 06 

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120131/94ac8740/attachment-0002.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: Postdoc_ad-FRM.pdf
Size: 140383 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120131/94ac8740/Postdoc_ad-FRM-0001.pdf>
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120131/94ac8740/attachment-0003.htm>

From mah79 at cam.ac.uk  Wed Feb  1 11:45:16 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Wed, 1 Feb 2012 11:45:16 +0000 (GMT)
Subject: [Pombelist] PomBase web site update - Orfeome localisation links
Message-ID: <Pine.LNX.4.64.1202011132250.31266@hermes-2.csi.cam.ac.uk>

Dear all,

We are pleased to announce that the PomBase web site, www.pombase.org has 
been updated to ensure that links to the Orfeome localisation data at 
SPD/RIKEN work correctly. Many thanks to all users who alerted us to the 
problem.

Details of the display of GO annotations and curated orthologs have also 
been improved.

Please send any questions or comments on the Pombase web site to us at 
<helpdesk at pombase.org>.

Best regards,
Midori

Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk



From banerji0 at googlemail.com  Thu Feb  2 11:13:01 2012
From: banerji0 at googlemail.com (Taniya Banerji)
Date: Thu, 2 Feb 2012 16:43:01 +0530
Subject: [Pombelist] Haploids
Message-ID: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>

Dear pombelist community,

I am trying to construct a double deleted mutant strain. I started with the
single deleted mutant strains and mated them. After mating them I
sporulated the mated cells, then I repeatedly streaked the diploid cells on
selective plates to get haploids. Finally the cells grown on selective
plates were streaked on phloxin B plates,which showed light pink colonies.
And at the same time I did flow cytometry analysis with these double
deleted mutant cells along with a different haploid strain as control. The
mutant cell showed the same DNA content as the haploid cells.
Is this process enough for checking haploid cells? Or can you suggest any
other confirmatory experiment to check haploid cells?
Sushobhana Bandyopadhyay
Junior Research Fellow
University of Calcutta.
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/eeffd967/attachment.htm>

From hoffmacs at bc.edu  Thu Feb  2 16:07:10 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 2 Feb 2012 11:07:10 -0500
Subject: [Pombelist] Haploids
In-Reply-To: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
Message-ID: <E6CC256E-55C3-4EAF-97EB-A0833F63A5F1@bc.edu>

In general, S. pombe strains are not stable as diploids, so depending on what medium you used for your cross, you would have been unlikely to have many diploids after the mating.  While I am a big proponent of tetrad dissection, it seems that you probably have your double mutant haploid strain if it displays the phenotype associated with each deletion.

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"



On Feb 2, 2012, at 6:13 AM, Taniya Banerji wrote:

> Dear pombelist community,
> 
> I am trying to construct a double deleted mutant strain. I started with the single deleted mutant strains and mated them. After mating them I sporulated the mated cells, then I repeatedly streaked the diploid cells on selective plates to get haploids. Finally the cells grown on selective plates were streaked on phloxin B plates,which showed light pink colonies. And at the same time I did flow cytometry analysis with these double deleted mutant cells along with a different haploid strain as control. The mutant cell showed the same DNA content as the haploid cells.
> Is this process enough for checking haploid cells? Or can you suggest any other confirmatory experiment to check haploid cells?
> Sushobhana Bandyopadhyay
> Junior Research Fellow
> University of Calcutta.
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist






From hoffmacs at bc.edu  Thu Feb  2 16:14:12 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 2 Feb 2012 11:14:12 -0500
Subject: [Pombelist] More on pombe transformation (LiOAc-DMSO)
Message-ID: <83833A3C-6CC9-404A-A9A8-FD7D1AA0EE73@bc.edu>

Hi all,

Along with seeing that resuspending pombe in water before plating kills cells and reduces the number of transformants, we see that the time spent incubating with PEG/LiOAc has a big effect.

Here are our recent results from a timecourse of incubation.  Next week, I am having students look at whether the carrier DNA matters, whether the heat shock matters, whether the DMSO matters, and whether the DMSO should be added at the same time as the PEG rather than right before the heat shock.  I welcome all guesses as to whether or not each of these changes will increase the number of transformants, decrease the number, or have no effect.  

Time course    Number of colonies

30 min                    28
60 min                    84
120 min                100
180 min                140
240 min                524   (wow-is this reproducible?)
21 hours                 10        
24 hours                 26
76 hours                 24


Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"





From klara at mail.nih.gov  Thu Feb  2 16:19:32 2012
From: klara at mail.nih.gov (Amar)
Date: Thu, 2 Feb 2012 11:19:32 -0500
Subject: [Pombelist] Haploids
In-Reply-To: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
Message-ID: <p0602043fcb50658a3264@[129.43.18.12]>

It is a trivial problem to deal with. Just dissect a few tetrads, 
grow segregants, check their markers. You will get your double 
mutant. If you don't get it, may be double mutant is dead. It will 
help to know if your deletions are tagged with drug markers.

The way you are going about is indirect, problematic. How can you 
sporulate the mated guys, then keep streaking to select diploids. 
This is what S. cerevisiae people do, them get frustrated to conclude 
pombe is difficult organism and quit working on it. I know it as I 
have been a cereviisae person for years; see for example my short (7 
pages) attached humorous memoir on budding yeast mating type studies.

Just a thought. I hope my suggestion helps.

Amar

>Dear pombelist community,
>
>I am trying to construct a double deleted mutant strain. I started 
>with the single deleted mutant strains and mated them. After mating 
>them I sporulated the mated cells, then I repeatedly streaked the 
>diploid cells on selective plates to get haploids. Finally the cells 
>grown on selective plates were streaked on phloxin B plates,which 
>showed light pink colonies. And at the same time I did flow 
>cytometry analysis with these double deleted mutant cells along with 
>a different haploid strain as control. The mutant cell showed the 
>same DNA content as the haploid cells.
>Is this process enough for checking haploid cells? Or can you 
>suggest any other confirmatory experiment to check haploid cells?
>Sushobhana Bandyopadhyay
>Junior Research Fellow
>University of Calcutta.
>
>Content-Type: text/plain; name="ATT00001.txt"
>Content-Description: ATT00001.txt
>Content-Disposition: attachment; filename="ATT00001.txt"; size=213;
>	creation-date="Thu, 02 Feb 2012 16:00:40 GMT";
>	modification-date="Thu, 02 Feb 2012 16:00:40 GMT"
>
>Attachment converted: Amar HD:ATT00001 201.txt (TEXT/ttxt) (03C1F5D1)


-- 
Amar Klar Ph.D
Section Head
Gene Regulation and Chromosome Biology laboratory
National Cancer Institute at Frederick
7th Street, Ft. Detrick
P.O. Box B, Frederick, MD 21702
Phone 301 846 5916
Fax 301 846 6911
E-mail: klara at mail.nih.gov
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: Klar_memoir.pdf
Size: 1165085 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/a218b1a5/Klar_memoir-0001.pdf>

From petrova at embl.de  Thu Feb  2 17:03:57 2012
From: petrova at embl.de (Boryana Petrova)
Date: Thu, 2 Feb 2012 18:03:57 +0100
Subject: [Pombelist] library complementation
In-Reply-To: <p0602043fcb50658a3264@[129.43.18.12]>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
	<p0602043fcb50658a3264@[129.43.18.12]>
Message-ID: <084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>

Dear Pombe Community,

I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?

Any help would be greatly appreciated!

Best wishes,
Boryana Petrova
PhD Student
EMBL, Heidelberg

From hoffmacs at bc.edu  Thu Feb  2 17:27:51 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 2 Feb 2012 12:27:51 -0500
Subject: [Pombelist] library complementation
In-Reply-To: <084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
	<p0602043fcb50658a3264@[129.43.18.12]>
	<084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
Message-ID: <96AEE480-5391-4820-A983-E1D931B5D58D@bc.edu>

It might be worth it to add phloxine B (15 mg/liter) to your plates for the screening at restrictive temperature.  Many ts strains in pombe appear to grow enough that you see  a colony, but all the cells have died (which you cannot see unless the phloxine B is there).

Charlie


On Feb 2, 2012, at 12:03 PM, Boryana Petrova wrote:

> Dear Pombe Community,
> 
> I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?
> 
> Any help would be greatly appreciated!
> 
> Best wishes,
> Boryana Petrova
> PhD Student
> EMBL, Heidelberg
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"





From a.m.carr at sussex.ac.uk  Thu Feb  2 18:59:59 2012
From: a.m.carr at sussex.ac.uk (Antony Carr)
Date: Thu, 2 Feb 2012 18:59:59 +0000
Subject: [Pombelist] library complementation
In-Reply-To: <084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
	<p0602043fcb50658a3264@[129.43.18.12]>
	<084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
Message-ID: <C5AD4BE27ED4F042BF57EA254C30BC2228D08617@EX-SHA-MBX1.ad.susx.ac.uk>

Boryana

If you are complementing a Ts mutant, then transform the library into the mutant and plate onto -ura plates and grow at the permissive temperature (i.e. 27oC). 

To screen the library and be confident you can or cannot clone the gene of interest (ie not there, toxic etc) you will have to screen about 100,000 colonies. I found that between 1 and 2000 colonies per plate was perfectly acceptable, so you can use 50-100 plates before you give up.

Allow colonies to grow up for 4-7 days, then replica plate from this (1000 ish colonies per plate - see above) onto two YE (non selective) plates with phloxin. Incubate 1 plate at the permissive (27oC) temperature and 1 plate at the restrictive temperature (36oC) for 12-24 hours. Scan the restrictive (36oC) plate for the least red/pink colonies (i.e. those that grow best amongst their friends on the same plate). Compare the same colony on the 27oC plate. A successful complementation should be less obviously "better growing" compared to their friends (ie the surrounding colonies on the 27oC plate) than is obvious on the 36oC plate.

Pick these colonies and streak to single cells on fresh YE (non selective)plates. Grow at the permissive temp (27oC) for 4-7 days until you have lots of individual colonies. Now replica these plates to two new plates: 1 YE+ phloxin  to be incubated at the restrictive temperature (36oC) and the  second, a minimal - ura plate, to be incubated at the permissive temperature (27oC). If you have a completing clone, the original plate should have had a mix of two types of colony: Type 1: plasmid containing. I.e. these show growth on minus ura plate at 27oC and less red on the YE+Ploxin plate at the restrictive temperature (36oC). Type 2. Plasmid is lost. I.e. these show no growth on Min -ura at 27oC and are very red on the YE phloxin at 36oC). If you have an integration, or a plasmid that doesn't complement, this will be obvious because you will not have the two types as above.


Do NOT, whatever anybody advises you, waste time by trying to clone by directly transforming the library and plating on selective plates  and then incubating these at 36oC. While this can work if you are lucky, but my experience is that, for greater than 50% of things I have tried, this does not work, for a variety of reasons. Since doing it properly involves only a few extra plates and a few extra days, you risk wasting much more time than you can potentially save by being "lucky"

Finally, we have cloned over 30 genes from this library. I think in only one case did we have problems due to the gene not being there. This was a gene that was greater than 7kb in size.
 

Tony Carr
Director, GDSC
University of Sussex
01273 678122


-----Original Message-----
From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Boryana Petrova
Sent: 02 February 2012 17:04
To: pombelist at sanger.ac.uk
Subject: [Pombelist] library complementation

Dear Pombe Community,

I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?

Any help would be greatly appreciated!

Best wishes,
Boryana Petrova
PhD Student
EMBL, Heidelberg
_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From k.tomita at ucl.ac.uk  Thu Feb  2 23:01:55 2012
From: k.tomita at ucl.ac.uk (Kazunori Tomita)
Date: Thu, 2 Feb 2012 23:01:55 +0000
Subject: [Pombelist] A post-doc position at the University College London, UK
Message-ID: <CB50C663.2589%kazunori.tomita@cancer.ucl.ac.uk>

Dear pombe researchers,

I hope this will be the last announcement.
I am still looking for a motivated post-doc to conduct pombe meiosis projects.
The position is initially 3 years and can be extended.
I am particularly keen to hear from newly graduated post-docs who wish to investigate the mystery of the bouquet.  Expertise on fluorescent microscopy, particularly live cell imaging techniques is ideal.
Deadline is 23rd February.

I would be appreciate if you could bring this to the attention of talented students/post-docs or friends who might know such people. Please use the poster attached or links below.


http://www.nature.com/naturejobs/science/jobs/243115<http://www.nature.com/naturejobs/science/jobs/230150>

Best regards,
Kazu Tomita

-------------------------------------------------->
Kazunori Tomita
CR-UK Career Development Fellow
Chromosome Maintenance Group

UCL Cancer Institute
University College London
72 Huntley Street
London WC1E 6DD, UK
Kazunori.tomita at cancer.ucl.ac.uk<mailto:Kazunori.tomita at cancer.ucl.ac.uk>
Phone: +44-20-7679-0769
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/ab2b1182/attachment-0001.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: TomitaK_RA2-PosterAd.pdf
Size: 152430 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/ab2b1182/TomitaK_RA2-PosterAd-0001.pdf>

From k.tomita at ucl.ac.uk  Fri Feb  3 10:52:37 2012
From: k.tomita at ucl.ac.uk (Kazunori Tomita)
Date: Fri, 3 Feb 2012 10:52:37 +0000
Subject: [Pombelist] A post-doc position at the University College London, UK
Message-ID: <CB516C84.FD6A%kazunori.tomita@cancer.ucl.ac.uk>

Dear pombe researchers,

The link to nature job was not working so I am sending amended one.

I hope this will be the last announcement.
I am still looking for a motivated post-doc to conduct pombe meiosis projects.
The position is initially 3 years and can be extended.
I am particularly keen to hear from newly graduated post-docs who wish to investigate the mystery of the bouquet.  Expertise on fluorescent microscopy, particularly live cell imaging techniques is ideal.
Deadline is 23rd February.

I would be appreciate if you could bring this to the attention of talented students/post-docs or friends who might know such people. Please use the poster attached or links below.


http://www.nature.com/naturejobs/science/jobs/243115

For poster to download,
http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/ab2b1182/TomitaK_RA2-PosterAd.pdf

Best regards,
Kazu Tomita

-------------------------------------------------->
Kazunori Tomita
CR-UK Career Development Fellow
Chromosome Maintenance Group

UCL Cancer Institute
University College London
72 Huntley Street
London WC1E 6DD, UK
Kazunori.tomita at cancer.ucl.ac.uk<mailto:Kazunori.tomita at cancer.ucl.ac.uk>
Phone: +44-20-7679-0769
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120203/a948cccd/attachment-0001.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: TomitaK_RA2-PosterAd.pdf
Size: 152430 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120203/a948cccd/TomitaK_RA2-PosterAd-0001.pdf>

From bayfield at yorku.ca  Fri Feb  3 19:23:42 2012
From: bayfield at yorku.ca (Mark Bayfield)
Date: Fri, 3 Feb 2012 14:23:42 -0500
Subject: [Pombelist] human cDNA library for expression in pombe
Message-ID: <DB5B3F96-E81A-43C3-AAF4-65580779CD6A@yorku.ca>

Hello everyone,

This was already asked a few years ago but I'm hoping the situation is more promising now:  Is anyone aware of a human cDNA library for expression in pombe?  We're hoping to use a ura marker.

Best,
Mark Bayfield



__________________________________
Mark Bayfield
Assistant Professor, Department of Biology
York University
4700 Keele St.
Life Science Building Room 327E
Toronto, ON M3J 1P3
Canada 
voice: (416) 736-2100 x44085 (office) x 77685 (lab)
bayfield at yorku.ca

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120203/dbf7e867/attachment.htm>

From Julie.Cooper at cancer.org.uk  Fri Feb 10 13:54:08 2012
From: Julie.Cooper at cancer.org.uk (Julie Cooper)
Date: Fri, 10 Feb 2012 13:54:08 +0000
Subject: [Pombelist] Scientific officer position, Telomere Biology Lab,
 Cancer Research UK
Message-ID: <CB5AD200.15BD0%Julie.Cooper@cancer.org.uk>

Dear Pombe Colleagues,

A scientific officer position is available in the Telomere Biology Laboratory at the London Research Institute of Cancer Research UK.  We study the spectrum and mechanisms of telomere function through mitosis and meiosis using fission yeast as a model system.  This is a maternity-cover position with a 6-month fixed-term contract beginning in March, 2012, with a possibility of extension.  The role involves both lab management duties and basic research.  Salary is ?28,850-?33,350 per annum (inclusive of inner London allowance) depending on experience.  You must be an effective team player with a proven ability to perform basic research in a rigorous and organized manner, excellent interpersonal and analytical skills and a strong work ethic.

The link to the application can be found at
https://cruk.taleo.net/careersection/cruk_corporate/jobdetail.ftl?lang=en&job=SCI00189



Best wishes,
Julie Cooper




Julia Promisel Cooper, Ph.D.
Group Leader, Telomere Biology Laboratory
Cancer Research UK, London Research Institute
44 Lincoln?s Inn Fields
London WC2A 3LY, UK
Phone +44-207-269-3415
FAX +44-207-269-3258
Email julie.cooper at cancer.org.uk
Web http://www.london-research-institute.org.uk/research/78

[cid:3411726848_142393476]





NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered in England and Wales
Company Registered Number: 4325234.
Registered Charity Number: 1089464 and Scotland SC041666
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120210/594ab974/attachment-0001.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: image.png
Size: 55586 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120210/594ab974/image-0001.png>

From jinquanwen at xmu.edu.cn  Wed Feb 15 13:09:25 2012
From: jinquanwen at xmu.edu.cn (=?GBK?B?vfnIq87E?=)
Date: Wed, 15 Feb 2012 21:09:25 +0800 (CST)
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
Message-ID: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>

Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005




From agata_sm at yahoo.com  Wed Feb 15 20:29:27 2012
From: agata_sm at yahoo.com (Agata Smialowska)
Date: Wed, 15 Feb 2012 12:29:27 -0800 (PST)
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
	<31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
Message-ID: <1329337767.70827.YahooMailNeo@web121404.mail.ne1.yahoo.com>

Dear Quanwen,
I've faced similar problems with detection of high pI proteins on Western blot. My conditions for protein blotting are: 10mM CAPS pH10 (you need 11), 10% methanol, transfer: 100-150mA overnight in the cold room, with constant stirring. I think you may safely extend your transfer time. To improve the overall efficiency, I soak the gel?for 30 min?in the transfer buffer prior to transfer - to remove any SDS that might interfere with binding of proteins to the membrane.
Good luck!
Agata Smialowska

?


________________________________
 From: ??? <jinquanwen at xmu.edu.cn>
To: pombelist at sanger.ac.uk 
Sent: Wednesday, February 15, 2012 2:09 PM
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
 
Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120215/c7884e72/attachment.htm>

From benoit.arcangioli at pasteur.fr  Wed Feb 15 18:15:11 2012
From: benoit.arcangioli at pasteur.fr (Benoit Arcangioli)
Date: Wed, 15 Feb 2012 19:15:11 +0100
Subject: [Pombelist] EMBO course
Message-ID: <4F3BF62F.2020209@pasteur.fr>

Hello,
I would like to send to the list member the annoucement of the EMBO 
course on Fission yeast (1-13 July 2012).
I would like to know how many members are on the list to have an 
estimation of how deep will be the annoucement.
Thank you
Benoit Arcangioli


From smarcus at as.ua.edu  Wed Feb 15 21:34:51 2012
From: smarcus at as.ua.edu (Marcus, Steve)
Date: Wed, 15 Feb 2012 15:34:51 -0600
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
	<31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
Message-ID: <60FA3A54-9266-4218-B076-697D76EA877F@as.ua.edu>

Hi Quanwen,

We were able to detect Wee1-HA using the boiled protein extract preparation procedure described by Correa-Bordes and Nurse (Cell, 1995, 83:1001-1009). In this procedure, cells are boiled in RIPA buffer prior to breaking them with glass beads, which presumably kills most of the vacuolar protease activity that might otherwise hydrolyze susceptible proteins during the steps of lysate preparation.

Good luck!

Steve


--
Stevan Marcus, Ph.D.
Associate Professor
Director, Graduate Program in Biological Sciences
Co-Director, UA-Howard Hughes Medical Institute Undergraduate Research Program
The University of Alabama
3310 Science & Engineering Complex (SEC)
300 Hackberry Lane
Box 870344
Tuscaloosa, AL  35487

Tel: 205-348-8094
FAX: 205-348-1786
E-mail: smarcus at as.ua.edu<mailto:smarcus at as.ua.edu>

On Feb 15, 2012, at 7:09 AM, ??? wrote:

Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From smarcus at as.ua.edu  Wed Feb 15 21:27:31 2012
From: smarcus at as.ua.edu (Marcus, Steve)
Date: Wed, 15 Feb 2012 15:27:31 -0600
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
	<31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
Message-ID: <B8896450-1B59-48C4-92E9-A10FD8BA5B64@as.ua.edu>

Hi Quanwen,

We were able to detect Wee1-HA using the boiled protein extract preparation procedure described by Correa-Bordes and Nurse (Cell, 1995, 83:1001-1009). In this procedure, cells are boiled in RIPA buffer prior to breaking them with glass beads, which presumably kills most of the vacuolar protease activity that might otherwise hydrolyze susceptable proteins during the steps of lysate preparation.

Good luck!

Steve


--
Stevan Marcus, Ph.D.
Associate Professor
Director, Graduate Program in Biological Sciences
Co-Director, UA-Howard Hughes Medical Institute Undergraduate Research Program
The University of Alabama
3310 Science & Engineering Complex (SEC)
300 Hackberry Lane
Box 870344
Tuscaloosa, AL  35487

Tel: 205-348-8094
FAX: 205-348-1786
E-mail: smarcus at as.ua.edu<mailto:smarcus at as.ua.edu>

On Feb 15, 2012, at 7:09 AM, ?????? wrote:

Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?C3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From anne.daulny at irbbarcelona.org  Thu Feb 16 10:31:37 2012
From: anne.daulny at irbbarcelona.org (Anne Daulny)
Date: Thu, 16 Feb 2012 11:31:37 +0100
Subject: [Pombelist] temperature-sensitive exosome mutant
Message-ID: <CACvhwn5KtZvKTPebZqin19aWdFv6K=7+ZxucZ6VtSQHzop7B1A@mail.gmail.com>

Dear pombe community,

I am looking for a temperature-sensitive exosome mutant that would be
growing reasonably well at 30C but would be inhibited at higher
temperature. Does anybody have such a strain and would be willing to share
it with me?

Thanks a lot,

Anne
-- 
Postdoctoral Fellow
Institute for Research in Biomedicine (IRB)
Ferran Azorin's Lab
Parc Cient?fic de Barcelona
Baldiri Reixac 10-12
08028 Barcelona
Spain

Tel: (+34) 93 403 4963

Email: anne.daulny at irbbarcelona.org
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120216/62e69b79/attachment.htm>

From tolic at mpi-cbg.de  Fri Feb 17 16:48:23 2012
From: tolic at mpi-cbg.de (Iva Tolic-Norrelykke)
Date: Fri, 17 Feb 2012 17:48:23 +0100
Subject: [Pombelist] Postdoc position available: pombe biophysics
Message-ID: <6FA1F91B-C6C5-454F-A2E3-FFD54AF53ADE@mpi-cbg.de>

A post-doctoral position is avaliable in the laboratory of Iva Tolic- 
Norrelykke (http://www.mpi-cbg.de/research/research-groups/iva-tolic-norrelykke.html 
) at the Max Planck Institute of Molecular Cell Biology and Genetics  
in Dresden. We are interested in how motor proteins and microtubules  
self-organize to generate large-scale structures and movements in the  
cell. The main projects in the lab focus on the mechanism of nuclear  
oscillations that help chromosome pairing in meiosis, the mechanisms  
of kinetochore capture and spindle assembly, as well as on segregation  
of damaged proteins in dividing cells. Our approach is live cell  
imaging of microtubules and motors at the single-molecule level in  
fission yeast and mammalian cells, in combination with genetic and  
biophysical manipulations, e.g., laser ablation. We aim at  
quantitative descriptions and development of simple theoretical models  
that help us understand the self-organization processes in the cell.

We are seeking a highly motivated candidate for joining our  
interdisciplinary and international team. The applicant should have:
-  a PhD in Molecular Biology, Biochemistry or Physics, and less than  
3 years of relevant postdoctoral experience
-  at least one publication in a peer-reviewed journal
-  expertise in: molecular genetics and biochemistry (for biologists),  
or statistical physics, nonlinear dynamics, and simulations (for  
physicists).

Please send your application as a single PDF document containing a  
concise summary of previous work and research interests, a Curriculum  
Vitae, a publication list, and contact information of 2-3 potential  
referees to Iva Tolic-Norrelykke at tolic at mpi-cbg.de.



From seth.lilavivat at utah.edu  Thu Feb 16 00:13:18 2012
From: seth.lilavivat at utah.edu (Seth Lilavivat)
Date: Wed, 15 Feb 2012 17:13:18 -0700
Subject: [Pombelist] Chloroquine to inhibit proteases
Message-ID: <CAOfBKrD9QTH=5m2TZos36cFk+Wunk=FyiGXAKGU0FHO9P77wJA@mail.gmail.com>

I was wondering if anyone has ever used Chloroquine in their cultures
to inhibit lysosomal proteases / acidify lysosomal lumen. If so, what
concentration and method of treatment was ideal?

Thanks,
Seth


From lidia.vasilieva at bioch.ox.ac.uk  Tue Feb 21 12:41:01 2012
From: lidia.vasilieva at bioch.ox.ac.uk (Lidia Vasilieva)
Date: Tue, 21 Feb 2012 12:41:01 +0000
Subject: [Pombelist] RNA FISH
Message-ID: <BE8C4D3FFC7C2D49830176E9D80106958F0F939ECC@EXMBX04.ad.oak.ox.ac.uk>

Dear all,

We are trying to set up RNA FISH in pombe.  We are using cerevisiae protocol that used to work nicely with budding yeast but the same protocol doesn't seem to work in fission yeast. We either not getting a signal at all or getting too high background,  we suspect that it may have something to do with the efficiency of spheroplasting. 

We would really appreciate any advice on this and if you guys have a written protocol to share that would be very helpful! 

Thanks very much!
 
Regards,
Lidia

Lidia Vasiljeva, PhD
group leader
Wellcome Trust RCDF fellow,
Department of Biochemistry,
University of Oxford,
South Parks Road,
OX13QU
phone: (+44)1865-613352
lab page: http://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=675


________________________________________
From: pombelist-bounces at sanger.ac.uk [pombelist-bounces at sanger.ac.uk] On Behalf Of Antony Carr [a.m.carr at sussex.ac.uk]
Sent: Thursday, February 02, 2012 6:59 PM
To: Boryana Petrova; pombelist at sanger.ac.uk
Subject: Re: [Pombelist] library complementation

Boryana

If you are complementing a Ts mutant, then transform the library into the mutant and plate onto -ura plates and grow at the permissive temperature (i.e. 27oC).

To screen the library and be confident you can or cannot clone the gene of interest (ie not there, toxic etc) you will have to screen about 100,000 colonies. I found that between 1 and 2000 colonies per plate was perfectly acceptable, so you can use 50-100 plates before you give up.

Allow colonies to grow up for 4-7 days, then replica plate from this (1000 ish colonies per plate - see above) onto two YE (non selective) plates with phloxin. Incubate 1 plate at the permissive (27oC) temperature and 1 plate at the restrictive temperature (36oC) for 12-24 hours. Scan the restrictive (36oC) plate for the least red/pink colonies (i.e. those that grow best amongst their friends on the same plate). Compare the same colony on the 27oC plate. A successful complementation should be less obviously "better growing" compared to their friends (ie the surrounding colonies on the 27oC plate) than is obvious on the 36oC plate.

Pick these colonies and streak to single cells on fresh YE (non selective)plates. Grow at the permissive temp (27oC) for 4-7 days until you have lots of individual colonies. Now replica these plates to two new plates: 1 YE+ phloxin  to be incubated at the restrictive temperature (36oC) and the  second, a minimal - ura plate, to be incubated at the permissive temperature (27oC). If you have a completing clone, the original plate should have had a mix of two types of colony: Type 1: plasmid containing. I.e. these show growth on minus ura plate at 27oC and less red on the YE+Ploxin plate at the restrictive temperature (36oC). Type 2. Plasmid is lost. I.e. these show no growth on Min -ura at 27oC and are very red on the YE phloxin at 36oC). If you have an integration, or a plasmid that doesn't complement, this will be obvious because you will not have the two types as above.


Do NOT, whatever anybody advises you, waste time by trying to clone by directly transforming the library and plating on selective plates  and then incubating these at 36oC. While this can work if you are lucky, but my experience is that, for greater than 50% of things I have tried, this does not work, for a variety of reasons. Since doing it properly involves only a few extra plates and a few extra days, you risk wasting much more time than you can potentially save by being "lucky"

Finally, we have cloned over 30 genes from this library. I think in only one case did we have problems due to the gene not being there. This was a gene that was greater than 7kb in size.


Tony Carr
Director, GDSC
University of Sussex
01273 678122


-----Original Message-----
From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Boryana Petrova
Sent: 02 February 2012 17:04
To: pombelist at sanger.ac.uk
Subject: [Pombelist] library complementation

Dear Pombe Community,

I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?

Any help would be greatly appreciated!

Best wishes,
Boryana Petrova
PhD Student
EMBL, Heidelberg
_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

From sam31 at st-andrews.ac.uk  Thu Feb 23 14:16:53 2012
From: sam31 at st-andrews.ac.uk (Stuart MacNeill)
Date: Thu, 23 Feb 2012 14:16:53 +0000
Subject: [Pombelist] PhD position at the University of St Andrews for UK/EU
	student
Message-ID: <CB6BFACA.A067%sam31@st-andrews.ac.uk>

Dear All

Please forward the details of the opportunity below to anyone you think might be interested - a PDF poster for display is also attached.

----------------------------------

Fission yeast TFIIH: at the nexus of transcription and DNA repair

A PhD position is available at the University of St Andrews to study the biology of TFIIH complex in fission yeast. In this PhD project, available to any individual from the EU with a good first degree in the life sciences, you will build on recent progress in the reconstitution of TFIIH from fission yeast. Biochemical, genetic and structural studies of the complex and individual sub-complexes will be used to define their functions, mechanisms and interactions in DNA repair and transcription. For more information,  contact either of the project co-supervisors: Dr Stuart MacNeill (sam31 at st-and.ac.uk) or Prof Malcolm White (mfw2 at st-and.ac.uk).

----------------------------------

Many thanks and best wishes,

Stuart.

Dr Stuart A. MacNeill
Reader in Translational Biology
Biomedical Sciences Research Complex B306
University of St Andrews
North Haugh
St Andrews
FIFE KY16 9ST
UK

T: +44 1334 467 268
F: +44 1334 462 595
E: stuart.macneill at st-andrews.ac.uk
W: http://biology.st-andrews.ac.uk/macneill
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120223/66dbebe7/attachment-0001.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: TFIIH_PhD_2012.pdf
Size: 394313 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120223/66dbebe7/TFIIH_PhD_2012-0001.pdf>

From t.sideri at ucl.ac.uk  Tue Feb 28 17:22:58 2012
From: t.sideri at ucl.ac.uk (Sideri, Dora)
Date: Tue, 28 Feb 2012 17:22:58 +0000
Subject: [Pombelist] cell viability assay using FACS
Message-ID: <D965734944251044BC1FF97A820C440307C35C80@AMSPRD0104MB099.eurprd01.prod.exchangelabs.com>

Hello everyone,



I would like to assay cell viability using flow cytometry. I was wondering if anyone have some experience with this and could send me a protocol.



Many Thanks

Dora



--------------------------------------------------
Dora Sideri
Department of Genetics, Evolution and Environment
and UCL Cancer Institute
University College London
Darwin Building, Gower Street,
WC1E 6BT, London,UK
--------
 P: +44 (0)20 3108 1606
 www.bahlerlab.info<http://www.bahlerlab.info>
--------------------------------------------------
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120228/9cb92688/attachment.htm>

From luis.rokeach at umontreal.ca  Tue Feb 28 17:47:43 2012
From: luis.rokeach at umontreal.ca (Rokeach Luis)
Date: Tue, 28 Feb 2012 12:47:43 -0500
Subject: [Pombelist] cell viability assay using FACS
In-Reply-To: <D965734944251044BC1FF97A820C440307C35C80@AMSPRD0104MB099.eurprd01.prod.exchangelabs.com>
Message-ID: <CB727D6F.15677%luis.rokeach@umontreal.ca>

Hello Dora,

For reference, you can see the following of our publications.


Best,

Luis


 
Luis A. Rokeach, PhD
Professor
Department of Biochemistry
Universit? de Montr?al
C.P. 6128, succ. Centre-ville
Montr?al, Qc H3C 3J7 Canada
Phone 1(514) 343-6324; Fax 1(514) 343-2210
luis.rokeach at umontreal.ca
http://www.mapageweb.umontreal.ca/rokeach




Calnexin regulates apoptosis induced by inositol starvation in fission
yeast.
Gu?rin R, Beauregard PB, Leroux A, Rokeach LA.
PLoS One. 2009 Jul 16;4(7):e6244.

Pro-aging effects of glucose signaling through a G protein-coupled glucose
receptor in fission yeast.
Roux AE, Leroux A, Alaamery MA, Hoffman CS, Chartrand P, Ferbeyre G, Rokeach
LA.
PLoS Genet. 2009 Mar;5(3):e1000408. Epub 2009 Mar 6.

Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in
the fission yeast.
Gu?rin R, Arseneault G, Dumont S, Rokeach LA.
Mol Biol Cell. 2008 Oct;19(10):4404-20. Epub 2008 Aug 13.


Regulation of chronological aging in Schizosaccharomyces pombe by the
protein kinases Pka1 and Sck2.
Roux AE, Quissac A, Chartrand P, Ferbeyre G, Rokeach LA.
Aging Cell. 2006 Aug;5(4):345-57. Epub 2006 Jul 5.





Le 12-02-28 12:22, ??Sideri, Dora?? <t.sideri at ucl.ac.uk> a ?crit?:

> Hello everyone,
> 
>  
> 
> I would like to assay cell viability using flow cytometry. I was wondering if
> anyone have some experience with this and could send me a protocol.
> 
>  
> 
> Many Thanks
> 
> Dora
> 
>  
> 
> --------------------------------------------------
> Dora Sideri
> Department of Genetics, Evolution and Environment
> and UCL Cancer Institute
> University College London
> Darwin Building, Gower Street,
> WC1E 6BT, London,UK
> --------
>  P: +44 (0)20 3108 1606
>  www.bahlerlab.info <http://www.bahlerlab.info>
> --------------------------------------------------
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120228/539e4ec9/attachment.htm>

From clara_reis2002 at yahoo.co.uk  Tue Feb 28 19:16:25 2012
From: clara_reis2002 at yahoo.co.uk (Clara Reis)
Date: Tue, 28 Feb 2012 19:16:25 +0000 (GMT)
Subject: [Pombelist] pESP-1 or pESP-3 expression vectors
In-Reply-To: <CB727D6F.15677%luis.rokeach@umontreal.ca>
References: <D965734944251044BC1FF97A820C440307C35C80@AMSPRD0104MB099.eurprd01.prod.exchangelabs.com>
	<CB727D6F.15677%luis.rokeach@umontreal.ca>
Message-ID: <1330456585.9645.YahooMailNeo@web28602.mail.ukl.yahoo.com>

Hello everyone!

I am searching for pESP-1 vector ( Lu et al, 1997 Gene) or its derived pESP-3 (Stratagene). Unfortunately I don't find them commercially available.?
If anyone has them and is willing to send me a little I will be very grateful,?
Cheers!,
Clara

?
Clara C. Reis, PhD 
Telomere and Genome Stability Lab
Instituto Gulbenkian de Ci?ncia
Rua da Quinta Grande, 6
2780-156 Oeiras
Portugal
http://sites.igc.gulbenkian.pt/telomere/tgs/Welcome.html
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120228/c7f074e7/attachment.htm>

From pluskal at oist.jp  Fri Mar 16 05:10:55 2012
From: pluskal at oist.jp (Tomas Pluskal)
Date: Fri, 16 Mar 2012 05:10:55 +0000
Subject: [Pombelist] Phthalate in EMM2
Message-ID: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>

Dear pombe community,

I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.

The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.

In particular, I would like to know what is the role of the phthalate in the medium.

Best regards,

Tomas



[cid:2CE68A67-01E1-4823-BAEB-10D615E5F7C4 at oist.jp]


[cid:5E20FCD6-CBC0-40E1-9453-04D64C655346 at oist.jp]


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/d98c7be9/attachment-0001.htm>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: EMM2 Mitchison.png
Size: 128727 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/d98c7be9/EMM2Mitchison-0001.png>
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: EMM2 Nurse.png
Size: 254340 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/d98c7be9/EMM2Nurse-0001.png>

From Ken.Sawin at ed.ac.uk  Fri Mar 16 08:16:50 2012
From: Ken.Sawin at ed.ac.uk (Ken Sawin)
Date: Fri, 16 Mar 2012 08:16:50 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
Message-ID: <9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>

Dear Tomas,

I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!

Best wishes,

Ken


On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:

> Dear pombe community,
> 
> I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.
> 
> The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.
> 
> In particular, I would like to know what is the role of the phthalate in the medium.
> 
> Best regards,
> 
> Tomas 
> 
> 
> 
> <EMM2 Mitchison.png>
> 
> 
> <EMM2 Nurse.png>
> 
> 
> ===============================================
> Tomas Pluskal
> G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
> 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
> TEL:  +81-98-966-8684
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk






-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.



From paul.nurse at royalsociety.org  Fri Mar 16 08:44:59 2012
From: paul.nurse at royalsociety.org (Nurse, Paul)
Date: Fri, 16 Mar 2012 08:44:59 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
	<9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
Message-ID: <1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>

Ken you are exactly right. Paul N
On 16 Mar 2012, at 08:16, Ken Sawin wrote:

> Dear Tomas,
> 
> I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!
> 
> Best wishes,
> 
> Ken
> 
> 
> On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:
> 
>> Dear pombe community,
>> 
>> I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.
>> 
>> The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.
>> 
>> In particular, I would like to know what is the role of the phthalate in the medium.
>> 
>> Best regards,
>> 
>> Tomas 
>> 
>> 
>> 
>> <EMM2 Mitchison.png>
>> 
>> 
>> <EMM2 Nurse.png>
>> 
>> 
>> ===============================================
>> Tomas Pluskal
>> G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
>> 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
>> TEL:  +81-98-966-8684
>> 
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://lists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> Kenneth E. Sawin, Ph.D.
> The Wellcome Trust Centre for Cell Biology
> School of Biological Sciences, University of Edinburgh
> Swann Building, Mayfield Road
> Edinburgh EH9  3JR
> United Kingdom
> 
> tel: 44-131-650-7064
> fax: 44-131-650-7360
> email: ken.sawin at ed.ac.uk
> 
> 
> 
> 
> 
> 
> -- 
> The University of Edinburgh is a charitable body, registered in
> Scotland, with registration number SC005336.
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom. 

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org

Registered charity no. 207043


From pluskal at oist.jp  Fri Mar 16 08:54:37 2012
From: pluskal at oist.jp (Tomas Pluskal)
Date: Fri, 16 Mar 2012 08:54:37 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
	<9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
	<1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>
Message-ID: <8398F6FE-EB90-435A-8149-1CEB51D4893C@oist.jp>

Dear Ken & Paul,

Thank you for the answer!

I suppose the reason for including the acetate or phthalate is to maintain the pH, is that correct?


Tomas


On Mar 16, 2012, at 5:44 PM, Nurse, Paul wrote:

Ken you are exactly right. Paul N
On 16 Mar 2012, at 08:16, Ken Sawin wrote:

Dear Tomas,

I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!

Best wishes,

Ken


On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:

Dear pombe community,

I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.

The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.

In particular, I would like to know what is the role of the phthalate in the medium.

Best regards,

Tomas



<EMM2 Mitchison.png>


<EMM2 Nurse.png>


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk<mailto:ken.sawin at ed.ac.uk>






--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom.

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org<mailto:ithelpdesk at royalsociety.org>

Registered charity no. 207043

===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/fc15436a/attachment.htm>

From paul.nurse at royalsociety.org  Fri Mar 16 09:05:59 2012
From: paul.nurse at royalsociety.org (Nurse, Paul)
Date: Fri, 16 Mar 2012 09:05:59 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <8398F6FE-EB90-435A-8149-1CEB51D4893C@oist.jp>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
	<9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
	<1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>
	<8398F6FE-EB90-435A-8149-1CEB51D4893C@oist.jp>
Message-ID: <0DF7D88A-6341-440E-A1AE-5FE39CA5F53F@royalsociety.org>

Yes P
On 16 Mar 2012, at 08:54, Tomas Pluskal wrote:

Dear Ken & Paul,

Thank you for the answer!

I suppose the reason for including the acetate or phthalate is to maintain the pH, is that correct?


Tomas


On Mar 16, 2012, at 5:44 PM, Nurse, Paul wrote:

Ken you are exactly right. Paul N
On 16 Mar 2012, at 08:16, Ken Sawin wrote:

Dear Tomas,

I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!

Best wishes,

Ken


On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:

Dear pombe community,

I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.

The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.

In particular, I would like to know what is the role of the phthalate in the medium.

Best regards,

Tomas



<EMM2 Mitchison.png>


<EMM2 Nurse.png>


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk<mailto:ken.sawin at ed.ac.uk>






--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom.

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org<mailto:ithelpdesk at royalsociety.org>

Registered charity no. 207043

===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom. 

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org

Registered charity no. 207043


From benoit.arcangioli at pasteur.fr  Mon Mar 12 09:57:10 2012
From: benoit.arcangioli at pasteur.fr (Benoit Arcangioli)
Date: Mon, 12 Mar 2012 10:57:10 +0100
Subject: [Pombelist] EMBO Course on Fission yeast 1-13th July
Message-ID: <4F5DC876.1060603@pasteur.fr>

Dear all,
I'm please to inform you that the EMBO course on fission Yeast is open 
for registration:
http://events.embo.org/12-fission-yeast
Cheers
Iain Hagan
Olaf Nielsen
Benoit Arcangioli
-------------- Attachments --------------
To retrieve the following attachment, use the link provided
Name: Poster embo-fission yeast.psd
Size: 7929903 bytes
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120312/93d05597/Posterembo-fissionyeast-0001.psd>

From geetanjali.sundaram at gmail.com  Wed Mar 21 11:09:25 2012
From: geetanjali.sundaram at gmail.com (Geetanjali Sundaram)
Date: Wed, 21 Mar 2012 06:09:25 -0500
Subject: [Pombelist] EMM and G418
Message-ID: <CAMKSyE4Eyf0+QXiLAW9gcUg7Ug=fEX2R096Y2UC3KiF2Ub9Yuw@mail.gmail.com>

Dear pombelist members,
can anyone tell me whether G418 works well in EMM-Ura media. i have
been trying to make a mutant for which i do the selection on this
media. but when i reinoculate the cells selected on EMM-Ura+G418 into
YES+G418, they dont grow,while they do grow on EMM-Ura media.
Regards
Geetanjali
-- 
Geetanjali Sundaram
Assistant Professor
Department of Biochemistry
Ballygunje Science College
University of Calcutta
35 Ballygunje Circular Road
Kolkata-700019
Ph-9433191982


From esmaakkaya at yahoo.com  Wed Mar 21 11:53:13 2012
From: esmaakkaya at yahoo.com (Esma AKKAYA)
Date: Wed, 21 Mar 2012 04:53:13 -0700 (PDT)
Subject: [Pombelist] EMM and G418
In-Reply-To: <CAMKSyE4Eyf0+QXiLAW9gcUg7Ug=fEX2R096Y2UC3KiF2Ub9Yuw@mail.gmail.com>
References: <CAMKSyE4Eyf0+QXiLAW9gcUg7Ug=fEX2R096Y2UC3KiF2Ub9Yuw@mail.gmail.com>
Message-ID: <1332330793.34764.YahooMailNeo@web112517.mail.gq1.yahoo.com>

Dear Dr Sundaram,
?
I recently tried EMM+G418 to make sure if it works or not. I compared my strains on EMM only and EMM+G418.?Unfortunately G418?didn't work at all on EMM for my strains. The reason?may be?that the amonium chloride in EMM media is competing with the drug so you need to substitute amonium with something else. This is also well explained in Dr. Forsburg's PombeNet page (http://www-bcf.usc.edu/~forsburg/drugs.html). PMG media is suggested instead of EMM for G418.
?
PMG recipe: http://www-bcf.usc.edu/~forsburg/media.html#PMG
?
I haven't tried yet but in your case you may have a problem with ura- strains?because it is also added as a bonus on PombeNet page where they say Ura- cells grow more similarly to Ura+ cells on PMG. It may work if you plate your cells first on YES+G418 and then replicate them on EMM-ura. 
?
Best Regards,
?
Esma Ucisik-Akkaya
?
?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Esma Ucisik-Akkaya
Grad student?- NEIMAN LAB
Graduate Program in Molecular Genetics & Microbiology
Stony Brook University
323?Life Sciences Building
Stony Brook, NY 11794-5222
?
Lab phone: 1-(631)-632-1421
Lab page: http://www.stonybrook.edu/biochem/neiman/lab.html


________________________________
From: Geetanjali Sundaram <geetanjali.sundaram at gmail.com>
To: pombelist at sanger.ac.uk 
Sent: Wednesday, March 21, 2012 7:09 AM
Subject: [Pombelist] EMM and G418

Dear pombelist members,
can anyone tell me whether G418 works well in EMM-Ura media. i have
been trying to make a mutant for which i do the selection on this
media. but when i reinoculate the cells selected on EMM-Ura+G418 into
YES+G418, they dont grow,while they do grow on EMM-Ura media.
Regards
Geetanjali
-- 
Geetanjali Sundaram
Assistant Professor
Department of Biochemistry
Ballygunje Science College
University of Calcutta
35 Ballygunje Circular Road
Kolkata-700019
Ph-9433191982

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120321/f9d4ff4d/attachment.htm>

From daniel.scott at path.ox.ac.uk  Fri Mar 30 17:02:14 2012
From: daniel.scott at path.ox.ac.uk (Daniel Scott)
Date: Fri, 30 Mar 2012 17:02:14 +0100
Subject: [Pombelist] ade6 transcription start site
In-Reply-To: <4F13F992.2010408@sanger.ac.uk>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
	<4F13F992.2010408@sanger.ac.uk>
Message-ID: <CANbSe_ky3qu4prBT+QYcOByX+zgNLbDXS6hL8T6FTY_WOhKQPA@mail.gmail.com>

Hi there fellow pombologists,

A quick query regarding auxotrophic supplements to minimal media - we've
been looking at using adenine sulphate rather than the standard adenine
hydrochloride and wonder if anyone (a) has any experience of this and (b)
if there's any reason why it may not work.  The standard salt solution
added to minimal media does contain Na2SO4 at 14.1mM final concentration
which would only increase to ~15mM, so shouldn't be a huge change in the
anion concentration.  Looking forward to hearing of your sage insights!

Regards,

Dan Scott
Norbury Lab
Sir William Dunn School of Pathology
University of Oxford
-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120330/5f92012b/attachment.htm>

From ozan.aygun at nih.gov  Tue Apr  3 03:04:19 2012
From: ozan.aygun at nih.gov (Aygun, Ozan (NIH/NCI) [F])
Date: Mon, 2 Apr 2012 22:04:19 -0400
Subject: [Pombelist] Comprehensive promoter resource for S.pombe
Message-ID: <CB9FD2E3.2102%ayguno@mail.nih.gov>

Dear Pombe Investigators,

I am wondering if any of you can recommend me a research article data set or a website directory that compiled a comprehensive list of defined S.pombe promoter sequences? There is a database for S.cerevisiae promoters, but I was unable to locate something analogous for fission yeast.

Sincerely,

Ozan Aygun


**************************************
Ozan Aygun, Ph.D.

National Cancer Institute
National Institutes of Health
Bethesda, Maryland 20892-4260
USA

Tel: 301-594-5607
E-mail: ozan.aygun at nih.gov
**************************************




From mah79 at cam.ac.uk  Tue Apr  3 14:09:02 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Tue, 3 Apr 2012 14:09:02 +0100 (BST)
Subject: [Pombelist] Comprehensive promoter resource for S.pombe
In-Reply-To: <CB9FD2E3.2102%ayguno@mail.nih.gov>
References: <CB9FD2E3.2102%ayguno@mail.nih.gov>
Message-ID: <Pine.LNX.4.64.1204031403350.10563@hermes-2.csi.cam.ac.uk>

Dear Dr. Ozan Aygun,

We do not have promoter sequences annotated in PomBase (nor its 
predecessor GeneDB), and I don't know of a database for S. pombe 
equivalent to the S. cerevisiae SCPD.

The closest thing we have at present is a curated list of DNA binding 
consensus sequences, most of which are promoter elements such as 
transcription factor binding sites; we have recently made these available 
on a web page:

http://www.pombase.org/dna-binding-sites

I hope this page is of some use even though it's not exactly what you're 
asking for. We would be very happy to host or link to promoter data for 
pombe if anyone in the community can make such data available, or knows of 
a relevant resource. We also welcome specific additions or corrections to 
the DNA binding site page.

Best regards,
Midori

Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk


On Mon, 2 Apr 2012, Aygun, Ozan (NIH/NCI) [F] wrote:

> Dear Pombe Investigators,
>
> I am wondering if any of you can recommend me a research article data 
> set or a website directory that compiled a comprehensive list of defined 
> S.pombe promoter sequences? There is a database for S.cerevisiae 
> promoters, but I was unable to locate something analogous for fission 
> yeast.
>
> Sincerely,
>
> Ozan Aygun
>
>
> **************************************
> Ozan Aygun, Ph.D.
>
> National Cancer Institute
> National Institutes of Health
> Bethesda, Maryland 20892-4260
> USA
>
> Tel: 301-594-5607
> E-mail: ozan.aygun at nih.gov
> **************************************
>
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist
>


From j.bahler at ucl.ac.uk  Mon Apr 16 19:52:01 2012
From: j.bahler at ucl.ac.uk (Bahler, Jurg)
Date: Mon, 16 Apr 2012 18:52:01 +0000
Subject: [Pombelist] Tool to predict protein-protein interactions
Message-ID: <74249C6FD1C7CB4CB0E1451F04F8D194E045BA@DB3PRD0104MB132.eurprd01.prod.exchangelabs.com>

PInt (Pombe Interactome) is a tool that allows you to retrieve predictions of S. pombe protein interactions, based on more than
100 features. The predictions were generated using two machine learning algorithms, and the confidence scores returned are used to rank interactions according to their probability.
PInt can be used for hypothesis generation and for exploring the interactome neighbourhoods of proteins of interest, which can be visualized through a user-friendly interface: http://www.bahlerlab.info/PInt/

For details see:
Pancaldi V, Sara? ?., Rallis C., McLean J.R., P?evorovsk? M., Gould K., Beyer A. and B?hler J. (2012). Predicting the fission yeast protein interaction network.
G3, 2(4), 453-467 (http://www.g3journal.org/content/2/4/453.abstract)

Enjoy,
-J?rg

---
J?rg B?hler
Professor of Systems Biology

University College London
Department of Genetics, Evolution & Environment
and UCL Cancer Institute
Darwin Building, Gower Street
London WC1E 6BT
U.K.

P: +44(0)20 3108 1602 (Internal x51602)
F: +44(0)20 7679 7096
E: j.bahler at ucl.ac.uk<mailto:j.bahler at ucl.ac.uk>
W: www.bahlerlab.info<http://www.sanger.ac.uk/PostGenomics/S_pombe/>

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120416/9e2cece4/attachment.htm>

From mah79 at cam.ac.uk  Tue Apr 17 16:00:48 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Tue, 17 Apr 2012 16:00:48 +0100 (BST)
Subject: [Pombelist] PomBase sequence download
Message-ID: <Pine.LNX.4.64.1204171428520.16854@hermes-2.csi.cam.ac.uk>

Dear Pombe Researchers,

We are pleased to announce that sequence downloads are now working 
properly from gene pages at www.pombase.org. We have also ensured that 
exon coordinates are listed in the correct order, and added CDS 
coordinates to the summary at the top of each gene page.

Many thanks to all of our users, especially those who have reported the 
issues that we have recently addressed. Please let us know if you have any 
problems with the new or fixed features, or if you have any other comments 
about PomBase.

Best regards,
Midori


Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk



From oliver at singerinstruments.com  Thu Apr 26 10:20:37 2012
From: oliver at singerinstruments.com (Ollie Thompson)
Date: Thu, 26 Apr 2012 09:20:37 +0000
Subject: [Pombelist] Scientific Advisor role with Singer Instruments (UK)
Message-ID: <C59C476009C0BB46840014103F60535737BBE55A@SINCOSERVER2.SINCOINTL.local>

Dear Pombe list members,



Position available: Scientific Advisor (South-West UK)



We are seeking a new member of the Singer Instruments team!



We're looking for an outgoing, motivated, and enthusiastic scientist with an interest in biotechnology hardware and applications, who wishes to keep in touch with laboratory life.



A strong background in science is required, ideally with post-graduate experience in yeast genetics, microbiology, or cell biology. Prior consultancy experience is not necessary as training will be given. What is required is a pleasant, friendly, outgoing personality...table-football experience is desirable!



The position involves a variety of day-to-day roles mixing science, marketing, R&D, sales, and accounts management, and includes:



* promoting and raising awareness of our existing products to new and existing customers
* liaising with existing and potential customers to understand their research needs
* participating in smart marketing strategies and campaigns
* seeking new areas of application for our instruments within industrial and academic research
* managing customer information and sales data
* advising on the strategic direction for new scientific devices and contributing to their design



The ideal candidate will have an interest in acquiring knowledge and skills in a commercial environment, as well as maintaining an awareness of scientific developments. There is a healthy travel budget, and the position will involve some travel to domestic and international scientific meetings to engage with active research scientists.



This position would suit someone who is seeking to apply their scientific expertise to practical biotechnology applications, whilst gaining business skills and experience.


Apply at http://bit.ly/SingerJobs

Dr Oliver Thompson | Scientific Advisor
SingerInstrument Co. Ltd.
A responsibility to science!
www.singerinstruments.co.uk<blocked::http://www.singerinstruments.co.uk/>
yeast at singerinst.co.uk<blocked::mailto:yeast at singerinst.co.uk>
T: +44 (0)1984 640226
F: +44 (0)1984 641166
M: +44 (0) 7972323822

Awesome: Sign up to our newsletter!<http://www.singerinst.co.uk/test/index.php?option=com_content&task=view&id=208&Itemid=884>

Boring: Read our smallprint<http://www.singerinst.co.uk/test/index.php?option=com_content&task=view&id=212>

-------------- Attachments --------------
An HTML attachment was scrubbed...
URL: <http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120426/96348e7f/attachment.htm>

From delivani at mpi-cbg.de  Mon Jan  9 13:25:55 2012
From: delivani at mpi-cbg.de (Petrina Delivani)
Date: Mon, 9 Jan 2012 14:25:55 +0100
Subject: [Pombelist] TetR plasmid
Message-ID: <04CAB867-EE57-429C-93C9-DC9687EAB221@mpi-cbg.de>

Hello pombe fans!

I would like to label pombe chromosomes with the tetO system. 
Does anybody have a plasmid containing the tetR-NLS-floursescence marker sequence? And would be happy to spare me some?

Thanks, Petrina.



Petrina Delivani, PhD
Tolic-Norrelykke-Lab
MPI-CBG, Dresden
Pfotenhauerstr. 108
01307 Dresden

delivani at mpi-cbg.de
Tel: 0049-351-210-2479
Fax: 0049-351-210-2020
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120109/8b0ac106/attachment-0002.htm>

From forsburg at usc.edu  Tue Jan 10 01:07:31 2012
From: forsburg at usc.edu (SLForsburg)
Date: Mon, 9 Jan 2012 17:07:31 -0800
Subject: [Pombelist] Looking for inert genomic locus
Message-ID: <ACE7D910-CBCD-4F9C-A095-4027E0AA8954@usc.edu>

We want to use an inert sequence for a chromatin immunoprecipitation experiment.  It must be in the euchromatin, distant from replication origins,  and not transcribed (no genes, no ncRNA).  It's probably the most boring part of the genome.   It would be an added bonus if anyone has designed ChIP primers for it.  

Any suggestions?  I will summarize and post.

Thanks!

Susan F.

PS:  the pombe shop at cafepress has a new design:  the pombe international tour tee-shirt!

http://www.cafepress.com/+pombe_international_tour_white,606010033


 
{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120109/aecb5908/attachment-0002.htm>

From burcinaltun at gmail.com  Wed Jan 11 14:46:16 2012
From: burcinaltun at gmail.com (burcin altun)
Date: Wed, 11 Jan 2012 15:46:16 +0100
Subject: [Pombelist] (no subject)
Message-ID: <CA+5fPiOZX6jzs0K0tNw6VgK7cqW1gaG29M8kxyCDgPZXH7DFbg@mail.gmail.com>

Dear Pombe People,
I would like to inform about YAC clonning, As i know people use yac just
for big size inserts, but i m planning to use it just for 3genes and
totally my insert will be 1000nukleotid. Is there anyone who knows anything
about clonning small size inside YAC? Is it stable?
Thanks in advance
-- 

Burcin ALTUN
PhD Student
Yeast Molecular Genetics Group
International Centre for Genetic Engineering and Biotechnology (ICGEB)
Padriciano 99, 34012 Trieste, Italy
E-mail:   altun at icgeb.org <passos at icgeb.org>
Phone:  +39-340-8613728



*
*
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120111/7d22dded/attachment-0002.htm>

From burcinaltun at gmail.com  Wed Jan 11 16:23:43 2012
From: burcinaltun at gmail.com (burcin altun)
Date: Wed, 11 Jan 2012 17:23:43 +0100
Subject: [Pombelist] YAC clonning
Message-ID: <CA+5fPiMQXupyctUEGHdNG46ufsCaUR+QRAXV+DWe8LNe=nBTzA@mail.gmail.com>

Dear Pombe People,
I would like to inform about YAC clonning, As i know people use yac just
for big size inserts, but i m planning to use it just for 3genes and
totally my insert will be 1000nukleotid. Is there anyone who knows anything
about clonning small size inside YAC? Is it stable?
Thanks in advance

-- 

Burcin ALTUN
PhD Student
Yeast Molecular Genetics Group
International Centre for Genetic Engineering and Biotechnology (ICGEB)
Padriciano 99, 34012 Trieste, Italy
E-mail: altun at icgeb.org
Phone:  +39-340-8613728



*
*
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120111/70d36f3a/attachment-0002.htm>

From sponberg1 at gmail.com  Fri Jan 13 13:39:40 2012
From: sponberg1 at gmail.com (=?ISO-8859-1?Q?Bj=F8rn_sponberg?=)
Date: Fri, 13 Jan 2012 14:39:40 +0100
Subject: [Pombelist] List of all Convergent Gene pairs in S. Pombe
Message-ID: <CAHgjbtRAL=sokSQPgM7_1U4=QztS+XUvZHJNtQ2C-nwo-FizLA@mail.gmail.com>

Hi all,
I have read much about Convergent Gene pairs in S. Pombe. They should make
up about ~30% of all protein coding genes. Does anyone know of a list that
contain CG exclusively?
Thanks!


Cheers
Bj?rn

Bj?rn Sponberg M.Sc.
Department of Biochemistry and Biophysics
Stockholm University
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/d5ab8c64/attachment-0002.htm>

From punitprasad at gmail.com  Fri Jan 13 14:03:21 2012
From: punitprasad at gmail.com (Punit Prasad)
Date: Fri, 13 Jan 2012 15:03:21 +0100
Subject: [Pombelist] Inducible system
Message-ID: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>

Hello,
I am looking for non-leaky inducible promoter system in pombe. Can I get
some suggestions from the experts.
Thanks in advance
Punit

-- 
*Punit Prasad*
Post Doctoral Fellow
Dept of Bioscience and Nutrition
Karolinska Institute
Stockholm
Sweden
http://www.bionut.ki.se/groups/kek/
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/66ae8639/attachment-0002.htm>

From hoffmacs at bc.edu  Fri Jan 13 14:11:30 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Fri, 13 Jan 2012 09:11:30 -0500
Subject: [Pombelist] Inducible system
In-Reply-To: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
References: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
Message-ID: <DA8C3520-A26B-4C61-986C-5996A5372540@bc.edu>

urg1 may be the best system, but you have to put your gene into the urg1 locus.

http://www.ncbi.nlm.nih.gov/pubmed/21664261


fbp1 is also very good in liquid culture, but since the promoter turns on in stationary phase, it is will come on in cells in a colony on a plate.  It will also be tighter in an integrated single copy than on a multicopy plasmid, but it does not have to be at the fbp1 locus. 

http://www.ncbi.nlm.nih.gov/pubmed/2558974

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"



On Jan 13, 2012, at 9:03 AM, Punit Prasad wrote:

> Hello,
> I am looking for non-leaky inducible promoter system in pombe. Can I get some suggestions from the experts. 
> Thanks in advance
> Punit
> 
> -- 
> Punit Prasad
> Post Doctoral Fellow
> Dept of Bioscience and Nutrition
> Karolinska Institute
> Stockholm
> Sweden
> http://www.bionut.ki.se/groups/kek/
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist



-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/fedb0861/attachment-0002.htm>

From a.m.carr at sussex.ac.uk  Fri Jan 13 16:09:23 2012
From: a.m.carr at sussex.ac.uk (Antony Carr)
Date: Fri, 13 Jan 2012 16:09:23 +0000
Subject: [Pombelist] Inducible system
In-Reply-To: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
References: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
Message-ID: <C5AD4BE27ED4F042BF57EA254C30BC2228CF0BD8@EX-SHA-MBX1.ad.susx.ac.uk>

Charlie is correct, you could consider urg1. We have a number of stains and plasmids to help described in the link he sent.

Also, we have adapted it to reduce expression levels (unpublished) and you are welcome to try that as well.

Tony

Tony Carr
Director, GDSC
University of Sussex
01273 678122

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Punit Prasad
Sent: 13 January 2012 14:03
To: pombelist at sanger.ac.uk
Subject: [Pombelist] Inducible system

Hello,
I am looking for non-leaky inducible promoter system in pombe. Can I get some suggestions from the experts.
Thanks in advance
Punit

--
Punit Prasad
Post Doctoral Fellow
Dept of Bioscience and Nutrition
Karolinska Institute
Stockholm
Sweden
http://www.bionut.ki.se/groups/kek/
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/83c18655/attachment-0002.htm>

From MEC at stowers.org  Fri Jan 13 18:11:48 2012
From: MEC at stowers.org (Cook, Malcolm)
Date: Fri, 13 Jan 2012 12:11:48 -0600
Subject: [Pombelist] List of all Convergent Gene pairs in S. Pombe
In-Reply-To: <CAHgjbtRAL=sokSQPgM7_1U4=QztS+XUvZHJNtQ2C-nwo-FizLA@mail.gmail.com>
References: <CAHgjbtRAL=sokSQPgM7_1U4=QztS+XUvZHJNtQ2C-nwo-FizLA@mail.gmail.com>
Message-ID: <2C40E43D1F7A56408C4463FD245DDDF995638CD7@EXCHMB-02.stowers-institute.org>

Bjorn,

I have been building out genomic resources in S. Pombe in support of a research program, and have used this as an example to demo to a collaborator the value of using R/Bioconductor for posing such questions.

I hope you find informative the attached BED formatted file listing 2146 convergent pairs.  You should be able to open it in a text editor, excel, or upload it for display it at pombase or ensembl (http://fungi.ensembl.org/Schizosaccharomyces_pombe/)

Cheers,

~Malcolm

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Bj?rn sponberg
Sent: Friday, January 13, 2012 7:40 AM
To: pombelist at sanger.ac.uk
Subject: [Pombelist] List of all Convergent Gene pairs in S. Pombe

Hi all,
I have read much about Convergent Gene pairs in S. Pombe. They should make up about ~30% of all protein coding genes. Does anyone know of a list that contain CG exclusively?
Thanks!


Cheers
Bj?rn

Bj?rn Sponberg M.Sc.
Department of Biochemistry and Biophysics
Stockholm University
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/190f2667/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: pombeConvergentTx.bed
Type: application/octet-stream
Size: 168780 bytes
Desc: pombeConvergentTx.bed
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120113/190f2667/attachment.obj>

From bassem.alsady at gmail.com  Fri Jan 13 18:32:54 2012
From: bassem.alsady at gmail.com (Bassem Al-Sady)
Date: Fri, 13 Jan 2012 10:32:54 -0800
Subject: [Pombelist] pombe FPs
Message-ID: <4F1078D6.8030104@ucsf.edu>

Hi,

I am looking for fluorescent proteins that behave well in pombe for flow 
cytometry, esp. if anyone has tested the newer generation of orange and 
red proteins.

thank you,

Bassem

-- 
Bassem Al-Sady, Ph.D.
Postdoctoral Fellow
Department of Biochemistry and Biophysics
University of California, San Francisco
Genentech Hall, MC 2240
600 16th St, San Francisco CA 94158
1-415-514-4302 (lab)




From jag at imtech.res.in  Sat Jan 14 09:53:22 2012
From: jag at imtech.res.in (jag at imtech.res.in)
Date: Sat, 14 Jan 2012 15:23:22 +0530 (IST)
Subject: [Pombelist] ade6 transcription start site
Message-ID: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>

Hi:

We are intersted in manipulating ade6 gene but would like to be careful about
disrupting the transcription start and termination sites. Any information about
the location of these sites would be welcome.
Thanks
Jag

______________________________________________________________________
?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
?????? 39 ?, ???????? / Sector 39-A, Chandigarh
??? ???/PIN CODE :160036
??????/EPABX :0172 6665 201-202



From francis.stewart at biotec.tu-dresden.de  Sat Jan 14 20:08:18 2012
From: francis.stewart at biotec.tu-dresden.de (Francis Stewart)
Date: Sat, 14 Jan 2012 21:08:18 +0100
Subject: [Pombelist] Inducible system
In-Reply-To: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
References: <CAOm0vzi48kphp7=i1udPH5eUZ6Bm8Mwou8j7_awvJ+otkejcRQ@mail.gmail.com>
Message-ID: <68FB954E-F704-4F6B-BBB2-17017E47F218@biotec.tu-dresden.de>

Punit

you might like to look at the data in this paper, in particular Figure 2.

regards,

Francis



On 13 Jan 2012, at 15:03, Punit Prasad wrote:

> Hello,
> I am looking for non-leaky inducible promoter system in pombe. Can I get some suggestions from the experts. 
> Thanks in advance
> Punit
> 
> -- 
> Punit Prasad
> Post Doctoral Fellow
> Dept of Bioscience and Nutrition
> Karolinska Institute
> Stockholm
> Sweden
> http://www.bionut.ki.se/groups/kek/
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist


Prof. A. Francis Stewart,
Genomics, Technische Universitaet Dresden
BioInnovationZentrum
Tatzberg 47
01307 Dresden
tel +49-351-46340129 or 30
fax+49-351-46340143
http://www.biotec.tu-dresden.de/research-faculty/stewart/group-page/

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120114/a12ddce7/attachment-0004.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Erler Yeast.pdf
Type: application/pdf
Size: 285565 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120114/a12ddce7/attachment.pdf>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120114/a12ddce7/attachment-0005.htm>

From val at sanger.ac.uk  Mon Jan 16 10:18:58 2012
From: val at sanger.ac.uk (Val Wood)
Date: Mon, 16 Jan 2012 10:18:58 +0000
Subject: [Pombelist] ade6 transcription start site
In-Reply-To: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
Message-ID: <4F13F992.2010408@sanger.ac.uk>


Hello Jag,

The UTRS of ade6 appear to be short, and are represented in this view,
http://www.pombase.org/spombe/result/SPCC1322.13 as short "open boxes"
as derived from Rhind et al  transcriptome data PMID:21511999

// B?hler la transcriptome viewer for ade6
http://www.bahlerlab.info/cgi-bin/pombetv/pombetv?condition=13&genename=SPCC1322.13&action=display
and EST data indicates a slightly shorter transcript beginning at 
1316291 and ending at  1318035

I have updated the "consensus UTRs" to be based on these coordinates 
supported by ESTs FY124947 and FY087729

The information  used to derive the consensus UTRs will be displayed 
under the "Transcipt" section of the gene page shortly.

Val




On 14/01/2012 09:53, jag at imtech.res.in wrote:
> Hi:
>
> We are intersted in manipulating ade6 gene but would like to be careful about
> disrupting the transcription start and termination sites. Any information about
> the location of these sites would be welcome.
> Thanks
> Jag
>
> ______________________________________________________________________
> ?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
> Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
> ?????? 39 ?, ???????? / Sector 39-A, Chandigarh
> ??? ???/PIN CODE :160036
> ??????/EPABX :0172 6665 201-202
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120116/93033f1a/attachment-0002.htm>

From Nick.Rhind at umassmed.edu  Tue Jan 17 15:17:27 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Tue, 17 Jan 2012 15:17:27 +0000
Subject: [Pombelist] ade6 transcription start site
In-Reply-To: <4F13F992.2010408@sanger.ac.uk>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
	<4F13F992.2010408@sanger.ac.uk>
Message-ID: <544C3935-E0C9-478E-9C83-D6594F82FEA6@umassmed.edu>

Hi Jag,

Another resource you might want to investigate is an annotation of UTRs in Table S2 of Lantermann et al. <http://www.ncbi.nlm.nih.gov/pubmed/20118936>, based on the HybMap data from Dutrow et al. <http://www.ncbi.nlm.nih.gov/pubmed/18641648>.

Nick


On 16 Jan, 12, at 5:18 AM, Val Wood wrote:

> 
> Hello Jag,
> 
> The UTRS of ade6 appear to be short, and are represented in this view,
> http://www.pombase.org/spombe/result/SPCC1322.13 as short "open boxes"
> as derived from Rhind et al  transcriptome data PMID:21511999  
> 
> B?hler la transcriptome viewer for ade6
> http://www.bahlerlab.info/cgi-bin/pombetv/pombetv?condition=13&genename=SPCC1322.13&action=display
> and EST data indicates a slightly shorter transcript beginning at 1316291 and ending at  1318035
> 
> I have updated the "consensus UTRs" to be based on these coordinates supported by ESTs FY124947 and FY087729
> 
> The information  used to derive the consensus UTRs will be displayed under the "Transcipt" section of the gene page shortly.
> 
> Val
> 
> 
> 
> 
> On 14/01/2012 09:53, jag at imtech.res.in wrote:
>> Hi:
>> 
>> We are intersted in manipulating ade6 gene but would like to be careful about
>> disrupting the transcription start and termination sites. Any information about
>> the location of these sites would be welcome.
>> Thanks
>> Jag
>> 
>> ______________________________________________________________________
>> ?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
>> Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
>> ?????? 39 ?, ???????? / Sector 39-A, Chandigarh
>> ??? ???/PIN CODE :160036
>> ??????/EPABX :0172 6665 201-202
>> 
>> _______________________________________________
>> Pombelist mailing list
>> 
>> Pombelist at sanger.ac.uk
>> http://lists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From adele.marston at ed.ac.uk  Wed Jan 18 11:32:02 2012
From: adele.marston at ed.ac.uk (Adele Marston)
Date: Wed, 18 Jan 2012 11:32:02 +0000
Subject: [Pombelist] British Yeast Group Meeting - REGISTRATION IS OPEN
Message-ID: <304023AF-2CBB-486F-B22A-3F10C8DEDD37@ed.ac.uk>

Dear Pombe people,

REGISTRATION IS NOW OPEN for the 2012 BYG meeting in Edinburgh (21st-23rd March).

Costs are ?260 all inclusive for single occupancy room.
If you don't need accommodation, it's ?170 (all meals etc. are provided).
There is a discount for genetics society members.

Please register NOW at (<http://www.britishyeastgroup.org/>)
Abstracts should be sent to: byg2012 at britishyeastgroup.org (instructions are on the website) BEFORE 17 February.
The majority of the talks will be selected from the abstracts. If you do not wish to be considered for a talk, please let us know when you send your abstract.

We also have an exciting programme of invited speakers:

John Kilmartin (Keynote Lecture)
Jurg Bahler (Global gene expression)
Tony Carr (DNA damage)
Anne Donaldson (DNA replication)
Iain Hagan (Mitosis)
Jon Houseley (Non-coding RNA)
Ed Louis (Telomeres, genome evolution)
Carol Munro (Fungal pathogens)
Markus Ralser (Metabolic networks)
Ken Sawin (Microtubules)
Martin Singleton (Kinetochore structures)
Tomo Tanaka (Chromosome Segregation)

The meeting will take place in the University's conference centre. The conference space is large and open with plenty of space for posters and mingling over drinks. All accommodation is single occupancy (double room) with ensuite facilities. The venue is in a beautiful part of Edinburgh, close to Holyrood park and walking distance from the centre. 

As well as the conference dinner, we will have some traditional Scottish entertainment and have secured sufficient sponsorship to ensure a very entertaining meeting both scientifically and socially.

I look forward to seeing you in Edinburgh in March!

Adele

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120118/85b12c02/attachment-0002.htm>
-------------- next part --------------
An embedded and charset-unspecified text was scrubbed...
Name: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120118/85b12c02/attachment.txt>

From luis.rokeach at umontreal.ca  Thu Jan 19 18:52:21 2012
From: luis.rokeach at umontreal.ca (Rokeach Luis)
Date: Thu, 19 Jan 2012 18:52:21 -0000
Subject: [Pombelist] postdoc position
Message-ID: <C83F51F1.D388%luis.rokeach@umontreal.ca>

Dear Colleagues,
 
I would greatly appreciate your posting of this ad for a postdoc position in
my lab.
 
 


With best regards,
 

Luis
 
Luis A. Rokeach, PhD
Professor
Department of Biochemistry
Universit? de Montr?al
C.P. 6128, succ. Centre-ville
Phone 1(514) 343-6324; Fax 1(514) 343-2210
luis.rokeach at umontreal.ca <mailto:luis.rokeach at umontreal.ca>
http://mapageweb.umontreal.ca/rokeach
 
 
 


-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120119/dfb8b105/attachment-0001.htm>

From Pernilla.Bjerling at imbim.uu.se  Fri Jan 20 14:41:45 2012
From: Pernilla.Bjerling at imbim.uu.se (Pernilla Bjerling)
Date: Fri, 20 Jan 2012 15:41:45 +0100
Subject: [Pombelist] post-doc
Message-ID: <FE9E83FC-D326-4597-A2FB-CA87FB8D3CE5@imbim.uu.se>

A non-text attachment was scrubbed...
Name: Postdoc_epigenetics.doc
Type: application/msword
Size: 56320 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120120/a5d2b187/attachment.doc>
-------------- next part --------------


Dear All

I am hiring a post-doc (see the ad) AND a PhD student so please post  
and/or forward this to suitable candidates.

The best! / Pernilla

Pernilla Bjerling, PhD, Researcher
University of Uppsala
Dept. of Medical Biochemistry and Microbiology (IMBIM)
Box 582; SE-751 23 Uppsala; Sweden
Tel.: +46 18 471 6652 or +46 18 471 4243
Fax.: +46 18 471 4673
e-mail: pernilla.bjerling at imbim.uu.se
web page: http://www.imbim.uu.se/forskning/bjerlingresearch.html






From levinpombe at gmail.com  Tue Jan 24 18:56:47 2012
From: levinpombe at gmail.com (Levin Henry)
Date: Tue, 24 Jan 2012 13:56:47 -0500
Subject: [Pombelist] protocol for transforming 96 pombe strains at once
In-Reply-To: <4F13F992.2010408@sanger.ac.uk>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
	<4F13F992.2010408@sanger.ac.uk>
Message-ID: <0B299C4F-0880-43F4-AA4D-B3F5A683FA48@mail.nih.gov>

Hello All,
	We are trying to transform one plasmid into the Bioneer collection of 3000 haploid strains.  We have tried many of the protocols used to transform the S. cerevisiae deletion set and they are not working with sufficient efficiency.  I would appreciate hearing from anyone who has such a protocol. Thanks for your help.

Henry Levin
NIH, bld 18T, room 106




From drojass at med.uchile.cl  Wed Jan 25 20:23:46 2012
From: drojass at med.uchile.cl (Diego Rojas Soto)
Date: Wed, 25 Jan 2012 17:23:46 -0300 (CLST)
Subject: [Pombelist] pombe expression vector and strain
Message-ID: <65270.172.16.77.175.1327523026.squirrel@correo.med.uchile.cl>

Hello:

I would like express a transcription factor in pombe cells to study the
effect in ribosomal protein expression but the vector has a ura+ marker so
I need the pombe strain MP6-10B. Where can I get it?

Best regards


Diego Rojas Soto
University of Chile
Faculty of Medicine



From pei-yun.wu at univ-rennes1.fr  Tue Jan 31 12:20:47 2012
From: pei-yun.wu at univ-rennes1.fr (Jenny Wu)
Date: Tue, 31 Jan 2012 13:20:47 +0100
Subject: [Pombelist] Post-doctoral position
Message-ID: <E79194B3-0650-460C-97F2-698A0B8B581B@univ-rennes1.fr>

Hi everyone,

A post-doctoral position is available in my laboratory, the Genome Duplication and Maintenance group, at the Institute of Genetics and Development of Rennes (IGDR) in Brittany, France. Our team uses fission yeast to investigate the control of DNA replication and the role of the multiple layers regulating DNA synthesis in the maintenance of genome integrity. We are seeking candidates to conduct a project that will focus on the fundamental parameters that determine the organization and selection of replication origins. This position is supported by the French Foundation for Medical Research.

For further information regarding the position and the institute, please see the attached PDF file as well as the following website: 

http://igdr.univ-rennes1.fr/english/


Please contact me if you need any additional information, and thank you for your time.



Sincerely,

Jenny Wu

**************************************************************************
Laboratory of Genome Duplication and Maintenance
Institute of Genetics and Development of Rennes, CNRS UMR 6290
2 av du Professeur L?on Bernard
Rennes, France 35043
email: pei-yun.wu at univ-rennes1.fr
phone: +33 2 23 23 44 06 

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120131/94ac8740/attachment-0004.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Postdoc_ad-FRM.pdf
Type: application/pdf
Size: 140383 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120131/94ac8740/attachment.pdf>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120131/94ac8740/attachment-0005.htm>

From mah79 at cam.ac.uk  Wed Feb  1 11:45:16 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Wed, 1 Feb 2012 11:45:16 +0000 (GMT)
Subject: [Pombelist] PomBase web site update - Orfeome localisation links
Message-ID: <Pine.LNX.4.64.1202011132250.31266@hermes-2.csi.cam.ac.uk>

Dear all,

We are pleased to announce that the PomBase web site, www.pombase.org has 
been updated to ensure that links to the Orfeome localisation data at 
SPD/RIKEN work correctly. Many thanks to all users who alerted us to the 
problem.

Details of the display of GO annotations and curated orthologs have also 
been improved.

Please send any questions or comments on the Pombase web site to us at 
<helpdesk at pombase.org>.

Best regards,
Midori

Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk




From banerji0 at googlemail.com  Thu Feb  2 11:13:01 2012
From: banerji0 at googlemail.com (Taniya Banerji)
Date: Thu, 2 Feb 2012 16:43:01 +0530
Subject: [Pombelist] Haploids
Message-ID: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>

Dear pombelist community,

I am trying to construct a double deleted mutant strain. I started with the
single deleted mutant strains and mated them. After mating them I
sporulated the mated cells, then I repeatedly streaked the diploid cells on
selective plates to get haploids. Finally the cells grown on selective
plates were streaked on phloxin B plates,which showed light pink colonies.
And at the same time I did flow cytometry analysis with these double
deleted mutant cells along with a different haploid strain as control. The
mutant cell showed the same DNA content as the haploid cells.
Is this process enough for checking haploid cells? Or can you suggest any
other confirmatory experiment to check haploid cells?
Sushobhana Bandyopadhyay
Junior Research Fellow
University of Calcutta.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/eeffd967/attachment-0002.htm>

From hoffmacs at bc.edu  Thu Feb  2 16:07:10 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 2 Feb 2012 11:07:10 -0500
Subject: [Pombelist] Haploids
In-Reply-To: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
Message-ID: <E6CC256E-55C3-4EAF-97EB-A0833F63A5F1@bc.edu>

In general, S. pombe strains are not stable as diploids, so depending on what medium you used for your cross, you would have been unlikely to have many diploids after the mating.  While I am a big proponent of tetrad dissection, it seems that you probably have your double mutant haploid strain if it displays the phenotype associated with each deletion.

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"



On Feb 2, 2012, at 6:13 AM, Taniya Banerji wrote:

> Dear pombelist community,
> 
> I am trying to construct a double deleted mutant strain. I started with the single deleted mutant strains and mated them. After mating them I sporulated the mated cells, then I repeatedly streaked the diploid cells on selective plates to get haploids. Finally the cells grown on selective plates were streaked on phloxin B plates,which showed light pink colonies. And at the same time I did flow cytometry analysis with these double deleted mutant cells along with a different haploid strain as control. The mutant cell showed the same DNA content as the haploid cells.
> Is this process enough for checking haploid cells? Or can you suggest any other confirmatory experiment to check haploid cells?
> Sushobhana Bandyopadhyay
> Junior Research Fellow
> University of Calcutta.
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist







From hoffmacs at bc.edu  Thu Feb  2 16:14:12 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 2 Feb 2012 11:14:12 -0500
Subject: [Pombelist] More on pombe transformation (LiOAc-DMSO)
Message-ID: <83833A3C-6CC9-404A-A9A8-FD7D1AA0EE73@bc.edu>

Hi all,

Along with seeing that resuspending pombe in water before plating kills cells and reduces the number of transformants, we see that the time spent incubating with PEG/LiOAc has a big effect.

Here are our recent results from a timecourse of incubation.  Next week, I am having students look at whether the carrier DNA matters, whether the heat shock matters, whether the DMSO matters, and whether the DMSO should be added at the same time as the PEG rather than right before the heat shock.  I welcome all guesses as to whether or not each of these changes will increase the number of transformants, decrease the number, or have no effect.  

Time course    Number of colonies

30 min                    28
60 min                    84
120 min                100
180 min                140
240 min                524   (wow-is this reproducible?)
21 hours                 10        
24 hours                 26
76 hours                 24


Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"






From klara at mail.nih.gov  Thu Feb  2 16:19:32 2012
From: klara at mail.nih.gov (Amar)
Date: Thu, 2 Feb 2012 11:19:32 -0500
Subject: [Pombelist] Haploids
In-Reply-To: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
Message-ID: <p0602043fcb50658a3264@[129.43.18.12]>

It is a trivial problem to deal with. Just dissect a few tetrads, 
grow segregants, check their markers. You will get your double 
mutant. If you don't get it, may be double mutant is dead. It will 
help to know if your deletions are tagged with drug markers.

The way you are going about is indirect, problematic. How can you 
sporulate the mated guys, then keep streaking to select diploids. 
This is what S. cerevisiae people do, them get frustrated to conclude 
pombe is difficult organism and quit working on it. I know it as I 
have been a cereviisae person for years; see for example my short (7 
pages) attached humorous memoir on budding yeast mating type studies.

Just a thought. I hope my suggestion helps.

Amar

>Dear pombelist community,
>
>I am trying to construct a double deleted mutant strain. I started 
>with the single deleted mutant strains and mated them. After mating 
>them I sporulated the mated cells, then I repeatedly streaked the 
>diploid cells on selective plates to get haploids. Finally the cells 
>grown on selective plates were streaked on phloxin B plates,which 
>showed light pink colonies. And at the same time I did flow 
>cytometry analysis with these double deleted mutant cells along with 
>a different haploid strain as control. The mutant cell showed the 
>same DNA content as the haploid cells.
>Is this process enough for checking haploid cells? Or can you 
>suggest any other confirmatory experiment to check haploid cells?
>Sushobhana Bandyopadhyay
>Junior Research Fellow
>University of Calcutta.
>
>Content-Type: text/plain; name="ATT00001.txt"
>Content-Description: ATT00001.txt
>Content-Disposition: attachment; filename="ATT00001.txt"; size=213;
>	creation-date="Thu, 02 Feb 2012 16:00:40 GMT";
>	modification-date="Thu, 02 Feb 2012 16:00:40 GMT"
>
>Attachment converted: Amar HD:ATT00001 201.txt (TEXT/ttxt) (03C1F5D1)


-- 
Amar Klar Ph.D
Section Head
Gene Regulation and Chromosome Biology laboratory
National Cancer Institute at Frederick
7th Street, Ft. Detrick
P.O. Box B, Frederick, MD 21702
Phone 301 846 5916
Fax 301 846 6911
E-mail: klara at mail.nih.gov
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Klar_memoir.pdf
Type: application/pdf
Size: 1165085 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/a218b1a5/attachment.pdf>

From petrova at embl.de  Thu Feb  2 17:03:57 2012
From: petrova at embl.de (Boryana Petrova)
Date: Thu, 2 Feb 2012 18:03:57 +0100
Subject: [Pombelist] library complementation
In-Reply-To: <p0602043fcb50658a3264@[129.43.18.12]>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
	<p0602043fcb50658a3264@[129.43.18.12]>
Message-ID: <084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>

Dear Pombe Community,

I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?

Any help would be greatly appreciated!

Best wishes,
Boryana Petrova
PhD Student
EMBL, Heidelberg


From hoffmacs at bc.edu  Thu Feb  2 17:27:51 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 2 Feb 2012 12:27:51 -0500
Subject: [Pombelist] library complementation
In-Reply-To: <084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
	<p0602043fcb50658a3264@[129.43.18.12]>
	<084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
Message-ID: <96AEE480-5391-4820-A983-E1D931B5D58D@bc.edu>

It might be worth it to add phloxine B (15 mg/liter) to your plates for the screening at restrictive temperature.  Many ts strains in pombe appear to grow enough that you see  a colony, but all the cells have died (which you cannot see unless the phloxine B is there).

Charlie


On Feb 2, 2012, at 12:03 PM, Boryana Petrova wrote:

> Dear Pombe Community,
> 
> I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?
> 
> Any help would be greatly appreciated!
> 
> Best wishes,
> Boryana Petrova
> PhD Student
> EMBL, Heidelberg
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"






From a.m.carr at sussex.ac.uk  Thu Feb  2 18:59:59 2012
From: a.m.carr at sussex.ac.uk (Antony Carr)
Date: Thu, 2 Feb 2012 18:59:59 +0000
Subject: [Pombelist] library complementation
In-Reply-To: <084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
References: <CAK_C4oFEqVQ4x_+F+2XVfVDgK6W36sHCjhT0ZALZ5t3jS-1pOg@mail.gmail.com>
	<p0602043fcb50658a3264@[129.43.18.12]>
	<084692E5-FDB7-421B-B0B4-F51C95E513C5@embl.de>
Message-ID: <C5AD4BE27ED4F042BF57EA254C30BC2228D08617@EX-SHA-MBX1.ad.susx.ac.uk>

Boryana

If you are complementing a Ts mutant, then transform the library into the mutant and plate onto -ura plates and grow at the permissive temperature (i.e. 27oC). 

To screen the library and be confident you can or cannot clone the gene of interest (ie not there, toxic etc) you will have to screen about 100,000 colonies. I found that between 1 and 2000 colonies per plate was perfectly acceptable, so you can use 50-100 plates before you give up.

Allow colonies to grow up for 4-7 days, then replica plate from this (1000 ish colonies per plate - see above) onto two YE (non selective) plates with phloxin. Incubate 1 plate at the permissive (27oC) temperature and 1 plate at the restrictive temperature (36oC) for 12-24 hours. Scan the restrictive (36oC) plate for the least red/pink colonies (i.e. those that grow best amongst their friends on the same plate). Compare the same colony on the 27oC plate. A successful complementation should be less obviously "better growing" compared to their friends (ie the surrounding colonies on the 27oC plate) than is obvious on the 36oC plate.

Pick these colonies and streak to single cells on fresh YE (non selective)plates. Grow at the permissive temp (27oC) for 4-7 days until you have lots of individual colonies. Now replica these plates to two new plates: 1 YE+ phloxin  to be incubated at the restrictive temperature (36oC) and the  second, a minimal - ura plate, to be incubated at the permissive temperature (27oC). If you have a completing clone, the original plate should have had a mix of two types of colony: Type 1: plasmid containing. I.e. these show growth on minus ura plate at 27oC and less red on the YE+Ploxin plate at the restrictive temperature (36oC). Type 2. Plasmid is lost. I.e. these show no growth on Min -ura at 27oC and are very red on the YE phloxin at 36oC). If you have an integration, or a plasmid that doesn't complement, this will be obvious because you will not have the two types as above.


Do NOT, whatever anybody advises you, waste time by trying to clone by directly transforming the library and plating on selective plates  and then incubating these at 36oC. While this can work if you are lucky, but my experience is that, for greater than 50% of things I have tried, this does not work, for a variety of reasons. Since doing it properly involves only a few extra plates and a few extra days, you risk wasting much more time than you can potentially save by being "lucky"

Finally, we have cloned over 30 genes from this library. I think in only one case did we have problems due to the gene not being there. This was a gene that was greater than 7kb in size.
 

Tony Carr
Director, GDSC
University of Sussex
01273 678122


-----Original Message-----
From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Boryana Petrova
Sent: 02 February 2012 17:04
To: pombelist at sanger.ac.uk
Subject: [Pombelist] library complementation

Dear Pombe Community,

I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?

Any help would be greatly appreciated!

Best wishes,
Boryana Petrova
PhD Student
EMBL, Heidelberg
_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist



From k.tomita at ucl.ac.uk  Thu Feb  2 23:01:55 2012
From: k.tomita at ucl.ac.uk (Kazunori Tomita)
Date: Thu, 2 Feb 2012 23:01:55 +0000
Subject: [Pombelist] A post-doc position at the University College London, UK
Message-ID: <CB50C663.2589%kazunori.tomita@cancer.ucl.ac.uk>

Dear pombe researchers,

I hope this will be the last announcement.
I am still looking for a motivated post-doc to conduct pombe meiosis projects.
The position is initially 3 years and can be extended.
I am particularly keen to hear from newly graduated post-docs who wish to investigate the mystery of the bouquet.  Expertise on fluorescent microscopy, particularly live cell imaging techniques is ideal.
Deadline is 23rd February.

I would be appreciate if you could bring this to the attention of talented students/post-docs or friends who might know such people. Please use the poster attached or links below.


http://www.nature.com/naturejobs/science/jobs/243115<http://www.nature.com/naturejobs/science/jobs/230150>

Best regards,
Kazu Tomita

-------------------------------------------------->
Kazunori Tomita
CR-UK Career Development Fellow
Chromosome Maintenance Group

UCL Cancer Institute
University College London
72 Huntley Street
London WC1E 6DD, UK
Kazunori.tomita at cancer.ucl.ac.uk<mailto:Kazunori.tomita at cancer.ucl.ac.uk>
Phone: +44-20-7679-0769
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/ab2b1182/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: TomitaK_RA2-PosterAd.pdf
Type: application/pdf
Size: 152430 bytes
Desc: TomitaK_RA2-PosterAd.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/ab2b1182/attachment.pdf>

From k.tomita at ucl.ac.uk  Fri Feb  3 10:52:37 2012
From: k.tomita at ucl.ac.uk (Kazunori Tomita)
Date: Fri, 3 Feb 2012 10:52:37 +0000
Subject: [Pombelist] A post-doc position at the University College London, UK
Message-ID: <CB516C84.FD6A%kazunori.tomita@cancer.ucl.ac.uk>

Dear pombe researchers,

The link to nature job was not working so I am sending amended one.

I hope this will be the last announcement.
I am still looking for a motivated post-doc to conduct pombe meiosis projects.
The position is initially 3 years and can be extended.
I am particularly keen to hear from newly graduated post-docs who wish to investigate the mystery of the bouquet.  Expertise on fluorescent microscopy, particularly live cell imaging techniques is ideal.
Deadline is 23rd February.

I would be appreciate if you could bring this to the attention of talented students/post-docs or friends who might know such people. Please use the poster attached or links below.


http://www.nature.com/naturejobs/science/jobs/243115

For poster to download,
http://lists.sanger.ac.uk/pipermail/pombelist/attachments/20120202/ab2b1182/TomitaK_RA2-PosterAd.pdf

Best regards,
Kazu Tomita

-------------------------------------------------->
Kazunori Tomita
CR-UK Career Development Fellow
Chromosome Maintenance Group

UCL Cancer Institute
University College London
72 Huntley Street
London WC1E 6DD, UK
Kazunori.tomita at cancer.ucl.ac.uk<mailto:Kazunori.tomita at cancer.ucl.ac.uk>
Phone: +44-20-7679-0769
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120203/a948cccd/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: TomitaK_RA2-PosterAd.pdf
Type: application/pdf
Size: 152430 bytes
Desc: TomitaK_RA2-PosterAd.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120203/a948cccd/attachment.pdf>

From bayfield at yorku.ca  Fri Feb  3 19:23:42 2012
From: bayfield at yorku.ca (Mark Bayfield)
Date: Fri, 3 Feb 2012 14:23:42 -0500
Subject: [Pombelist] human cDNA library for expression in pombe
Message-ID: <DB5B3F96-E81A-43C3-AAF4-65580779CD6A@yorku.ca>

Hello everyone,

This was already asked a few years ago but I'm hoping the situation is more promising now:  Is anyone aware of a human cDNA library for expression in pombe?  We're hoping to use a ura marker.

Best,
Mark Bayfield



__________________________________
Mark Bayfield
Assistant Professor, Department of Biology
York University
4700 Keele St.
Life Science Building Room 327E
Toronto, ON M3J 1P3
Canada 
voice: (416) 736-2100 x44085 (office) x 77685 (lab)
bayfield at yorku.ca

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120203/dbf7e867/attachment-0002.htm>

From Julie.Cooper at cancer.org.uk  Fri Feb 10 13:54:08 2012
From: Julie.Cooper at cancer.org.uk (Julie Cooper)
Date: Fri, 10 Feb 2012 13:54:08 +0000
Subject: [Pombelist] Scientific officer position, Telomere Biology Lab,
 Cancer Research UK
Message-ID: <CB5AD200.15BD0%Julie.Cooper@cancer.org.uk>

Dear Pombe Colleagues,

A scientific officer position is available in the Telomere Biology Laboratory at the London Research Institute of Cancer Research UK.  We study the spectrum and mechanisms of telomere function through mitosis and meiosis using fission yeast as a model system.  This is a maternity-cover position with a 6-month fixed-term contract beginning in March, 2012, with a possibility of extension.  The role involves both lab management duties and basic research.  Salary is ?28,850-?33,350 per annum (inclusive of inner London allowance) depending on experience.  You must be an effective team player with a proven ability to perform basic research in a rigorous and organized manner, excellent interpersonal and analytical skills and a strong work ethic.

The link to the application can be found at
https://cruk.taleo.net/careersection/cruk_corporate/jobdetail.ftl?lang=en&job=SCI00189



Best wishes,
Julie Cooper




Julia Promisel Cooper, Ph.D.
Group Leader, Telomere Biology Laboratory
Cancer Research UK, London Research Institute
44 Lincoln?s Inn Fields
London WC2A 3LY, UK
Phone +44-207-269-3415
FAX +44-207-269-3258
Email julie.cooper at cancer.org.uk
Web http://www.london-research-institute.org.uk/research/78

[cid:3411726848_142393476]





NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered in England and Wales
Company Registered Number: 4325234.
Registered Charity Number: 1089464 and Scotland SC041666
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120210/594ab974/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: image.png
Type: image/png
Size: 55586 bytes
Desc: image.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120210/594ab974/attachment.png>

From jinquanwen at xmu.edu.cn  Wed Feb 15 13:09:25 2012
From: jinquanwen at xmu.edu.cn (=?GBK?B?vfnIq87E?=)
Date: Wed, 15 Feb 2012 21:09:25 +0800 (CST)
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
Message-ID: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>

Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005





From agata_sm at yahoo.com  Wed Feb 15 20:29:27 2012
From: agata_sm at yahoo.com (Agata Smialowska)
Date: Wed, 15 Feb 2012 12:29:27 -0800 (PST)
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
	<31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
Message-ID: <1329337767.70827.YahooMailNeo@web121404.mail.ne1.yahoo.com>

Dear Quanwen,
I've faced similar problems with detection of high pI proteins on Western blot. My conditions for protein blotting are: 10mM CAPS pH10 (you need 11), 10% methanol, transfer: 100-150mA overnight in the cold room, with constant stirring. I think you may safely extend your transfer time. To improve the overall efficiency, I soak the gel?for 30 min?in the transfer buffer prior to transfer - to remove any SDS that might interfere with binding of proteins to the membrane.
Good luck!
Agata Smialowska

?


________________________________
 From: ??? <jinquanwen at xmu.edu.cn>
To: pombelist at sanger.ac.uk 
Sent: Wednesday, February 15, 2012 2:09 PM
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
 
Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120215/c7884e72/attachment-0002.htm>

From benoit.arcangioli at pasteur.fr  Wed Feb 15 18:15:11 2012
From: benoit.arcangioli at pasteur.fr (Benoit Arcangioli)
Date: Wed, 15 Feb 2012 19:15:11 +0100
Subject: [Pombelist] EMBO course
Message-ID: <4F3BF62F.2020209@pasteur.fr>

Hello,
I would like to send to the list member the annoucement of the EMBO 
course on Fission yeast (1-13 July 2012).
I would like to know how many members are on the list to have an 
estimation of how deep will be the annoucement.
Thank you
Benoit Arcangioli



From smarcus at as.ua.edu  Wed Feb 15 21:34:51 2012
From: smarcus at as.ua.edu (Marcus, Steve)
Date: Wed, 15 Feb 2012 15:34:51 -0600
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
	<31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
Message-ID: <60FA3A54-9266-4218-B076-697D76EA877F@as.ua.edu>

Hi Quanwen,

We were able to detect Wee1-HA using the boiled protein extract preparation procedure described by Correa-Bordes and Nurse (Cell, 1995, 83:1001-1009). In this procedure, cells are boiled in RIPA buffer prior to breaking them with glass beads, which presumably kills most of the vacuolar protease activity that might otherwise hydrolyze susceptible proteins during the steps of lysate preparation.

Good luck!

Steve


--
Stevan Marcus, Ph.D.
Associate Professor
Director, Graduate Program in Biological Sciences
Co-Director, UA-Howard Hughes Medical Institute Undergraduate Research Program
The University of Alabama
3310 Science & Engineering Complex (SEC)
300 Hackberry Lane
Box 870344
Tuscaloosa, AL  35487

Tel: 205-348-8094
FAX: 205-348-1786
E-mail: smarcus at as.ua.edu<mailto:smarcus at as.ua.edu>

On Feb 15, 2012, at 7:09 AM, ??? wrote:

Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From smarcus at as.ua.edu  Wed Feb 15 21:27:31 2012
From: smarcus at as.ua.edu (Marcus, Steve)
Date: Wed, 15 Feb 2012 15:27:31 -0600
Subject: [Pombelist] problem to detect wee1-3HA-6His by Western
In-Reply-To: <31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
References: <mailman.1730.1328223780.29675.pombelist@sanger.ac.uk>
	<31493949.286551329311365179.JavaMail.coremail@mail.xmu.edu.cn>
Message-ID: <B8896450-1B59-48C4-92E9-A10FD8BA5B64@as.ua.edu>

Hi Quanwen,

We were able to detect Wee1-HA using the boiled protein extract preparation procedure described by Correa-Bordes and Nurse (Cell, 1995, 83:1001-1009). In this procedure, cells are boiled in RIPA buffer prior to breaking them with glass beads, which presumably kills most of the vacuolar protease activity that might otherwise hydrolyze susceptable proteins during the steps of lysate preparation.

Good luck!

Steve


--
Stevan Marcus, Ph.D.
Associate Professor
Director, Graduate Program in Biological Sciences
Co-Director, UA-Howard Hughes Medical Institute Undergraduate Research Program
The University of Alabama
3310 Science & Engineering Complex (SEC)
300 Hackberry Lane
Box 870344
Tuscaloosa, AL  35487

Tel: 205-348-8094
FAX: 205-348-1786
E-mail: smarcus at as.ua.edu<mailto:smarcus at as.ua.edu>

On Feb 15, 2012, at 7:09 AM, ?????? wrote:

Dear Pombe Community,

I have been trying to compare the endogenous Wee1 protein levels in Wild type and my mutant strains carrying wee1?C3HA-6His, but I have trouble to detect the Wee1-HA in Western, although my HA antibodies can recognize other HA-tagged proteins. I am not always seeing the expected bands (about 110kD), it seems the detection is not reproducible. I made yeast cell lysates using 2x Laemmli buffer with 8M Urea after breaking cells with glass beads. Since the isoelectric point for Wee1 is 10.2, I used CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid) buffer (pH 11.0) in tank tranfer onto PVDF membrane at 100V for 90-120min. I guess probably my transfer is not suffcient, does anyone have suggestions on how to improve my Western with Wee1? Thanks!

Quanwen

----------------------------
Quanwen Jin, Ph.D
School of Life Sciences
Xiamen University
Xiamen, Fujian
China 361005



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From anne.daulny at irbbarcelona.org  Thu Feb 16 10:31:37 2012
From: anne.daulny at irbbarcelona.org (Anne Daulny)
Date: Thu, 16 Feb 2012 11:31:37 +0100
Subject: [Pombelist] temperature-sensitive exosome mutant
Message-ID: <CACvhwn5KtZvKTPebZqin19aWdFv6K=7+ZxucZ6VtSQHzop7B1A@mail.gmail.com>

Dear pombe community,

I am looking for a temperature-sensitive exosome mutant that would be
growing reasonably well at 30C but would be inhibited at higher
temperature. Does anybody have such a strain and would be willing to share
it with me?

Thanks a lot,

Anne
-- 
Postdoctoral Fellow
Institute for Research in Biomedicine (IRB)
Ferran Azorin's Lab
Parc Cient?fic de Barcelona
Baldiri Reixac 10-12
08028 Barcelona
Spain

Tel: (+34) 93 403 4963

Email: anne.daulny at irbbarcelona.org
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120216/62e69b79/attachment-0002.htm>

From tolic at mpi-cbg.de  Fri Feb 17 16:48:23 2012
From: tolic at mpi-cbg.de (Iva Tolic-Norrelykke)
Date: Fri, 17 Feb 2012 17:48:23 +0100
Subject: [Pombelist] Postdoc position available: pombe biophysics
Message-ID: <6FA1F91B-C6C5-454F-A2E3-FFD54AF53ADE@mpi-cbg.de>

A post-doctoral position is avaliable in the laboratory of Iva Tolic- 
Norrelykke (http://www.mpi-cbg.de/research/research-groups/iva-tolic-norrelykke.html 
) at the Max Planck Institute of Molecular Cell Biology and Genetics  
in Dresden. We are interested in how motor proteins and microtubules  
self-organize to generate large-scale structures and movements in the  
cell. The main projects in the lab focus on the mechanism of nuclear  
oscillations that help chromosome pairing in meiosis, the mechanisms  
of kinetochore capture and spindle assembly, as well as on segregation  
of damaged proteins in dividing cells. Our approach is live cell  
imaging of microtubules and motors at the single-molecule level in  
fission yeast and mammalian cells, in combination with genetic and  
biophysical manipulations, e.g., laser ablation. We aim at  
quantitative descriptions and development of simple theoretical models  
that help us understand the self-organization processes in the cell.

We are seeking a highly motivated candidate for joining our  
interdisciplinary and international team. The applicant should have:
-  a PhD in Molecular Biology, Biochemistry or Physics, and less than  
3 years of relevant postdoctoral experience
-  at least one publication in a peer-reviewed journal
-  expertise in: molecular genetics and biochemistry (for biologists),  
or statistical physics, nonlinear dynamics, and simulations (for  
physicists).

Please send your application as a single PDF document containing a  
concise summary of previous work and research interests, a Curriculum  
Vitae, a publication list, and contact information of 2-3 potential  
referees to Iva Tolic-Norrelykke at tolic at mpi-cbg.de.




From seth.lilavivat at utah.edu  Thu Feb 16 00:13:18 2012
From: seth.lilavivat at utah.edu (Seth Lilavivat)
Date: Wed, 15 Feb 2012 17:13:18 -0700
Subject: [Pombelist] Chloroquine to inhibit proteases
Message-ID: <CAOfBKrD9QTH=5m2TZos36cFk+Wunk=FyiGXAKGU0FHO9P77wJA@mail.gmail.com>

I was wondering if anyone has ever used Chloroquine in their cultures
to inhibit lysosomal proteases / acidify lysosomal lumen. If so, what
concentration and method of treatment was ideal?

Thanks,
Seth



From lidia.vasilieva at bioch.ox.ac.uk  Tue Feb 21 12:41:01 2012
From: lidia.vasilieva at bioch.ox.ac.uk (Lidia Vasilieva)
Date: Tue, 21 Feb 2012 12:41:01 +0000
Subject: [Pombelist] RNA FISH
Message-ID: <BE8C4D3FFC7C2D49830176E9D80106958F0F939ECC@EXMBX04.ad.oak.ox.ac.uk>

Dear all,

We are trying to set up RNA FISH in pombe.  We are using cerevisiae protocol that used to work nicely with budding yeast but the same protocol doesn't seem to work in fission yeast. We either not getting a signal at all or getting too high background,  we suspect that it may have something to do with the efficiency of spheroplasting. 

We would really appreciate any advice on this and if you guys have a written protocol to share that would be very helpful! 

Thanks very much!
 
Regards,
Lidia

Lidia Vasiljeva, PhD
group leader
Wellcome Trust RCDF fellow,
Department of Biochemistry,
University of Oxford,
South Parks Road,
OX13QU
phone: (+44)1865-613352
lab page: http://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=675


________________________________________
From: pombelist-bounces at sanger.ac.uk [pombelist-bounces at sanger.ac.uk] On Behalf Of Antony Carr [a.m.carr at sussex.ac.uk]
Sent: Thursday, February 02, 2012 6:59 PM
To: Boryana Petrova; pombelist at sanger.ac.uk
Subject: Re: [Pombelist] library complementation

Boryana

If you are complementing a Ts mutant, then transform the library into the mutant and plate onto -ura plates and grow at the permissive temperature (i.e. 27oC).

To screen the library and be confident you can or cannot clone the gene of interest (ie not there, toxic etc) you will have to screen about 100,000 colonies. I found that between 1 and 2000 colonies per plate was perfectly acceptable, so you can use 50-100 plates before you give up.

Allow colonies to grow up for 4-7 days, then replica plate from this (1000 ish colonies per plate - see above) onto two YE (non selective) plates with phloxin. Incubate 1 plate at the permissive (27oC) temperature and 1 plate at the restrictive temperature (36oC) for 12-24 hours. Scan the restrictive (36oC) plate for the least red/pink colonies (i.e. those that grow best amongst their friends on the same plate). Compare the same colony on the 27oC plate. A successful complementation should be less obviously "better growing" compared to their friends (ie the surrounding colonies on the 27oC plate) than is obvious on the 36oC plate.

Pick these colonies and streak to single cells on fresh YE (non selective)plates. Grow at the permissive temp (27oC) for 4-7 days until you have lots of individual colonies. Now replica these plates to two new plates: 1 YE+ phloxin  to be incubated at the restrictive temperature (36oC) and the  second, a minimal - ura plate, to be incubated at the permissive temperature (27oC). If you have a completing clone, the original plate should have had a mix of two types of colony: Type 1: plasmid containing. I.e. these show growth on minus ura plate at 27oC and less red on the YE+Ploxin plate at the restrictive temperature (36oC). Type 2. Plasmid is lost. I.e. these show no growth on Min -ura at 27oC and are very red on the YE phloxin at 36oC). If you have an integration, or a plasmid that doesn't complement, this will be obvious because you will not have the two types as above.


Do NOT, whatever anybody advises you, waste time by trying to clone by directly transforming the library and plating on selective plates  and then incubating these at 36oC. While this can work if you are lucky, but my experience is that, for greater than 50% of things I have tried, this does not work, for a variety of reasons. Since doing it properly involves only a few extra plates and a few extra days, you risk wasting much more time than you can potentially save by being "lucky"

Finally, we have cloned over 30 genes from this library. I think in only one case did we have problems due to the gene not being there. This was a gene that was greater than 7kb in size.


Tony Carr
Director, GDSC
University of Sussex
01273 678122


-----Original Message-----
From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Boryana Petrova
Sent: 02 February 2012 17:04
To: pombelist at sanger.ac.uk
Subject: [Pombelist] library complementation

Dear Pombe Community,

I am currently trying to complement a temperature sensitive mutant with a genomic library (originally described by A. Carr; URA-selection based). I would be grateful for any suggestions. For example, after transformation with the library the appearing colonies are all different sizes, later it is very difficult to select a "better growing" one on the high temperature plates. Any suggestion to circumvent this? One other problem is that often a colony would grow better just because of a plasmid containing a genomic piece with a metabolic enzyme. Could one reduce this background? Also what should I expect, how many colonies is it reasonable to screen?

Any help would be greatly appreciated!

Best wishes,
Boryana Petrova
PhD Student
EMBL, Heidelberg
_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist


From sam31 at st-andrews.ac.uk  Thu Feb 23 14:16:53 2012
From: sam31 at st-andrews.ac.uk (Stuart MacNeill)
Date: Thu, 23 Feb 2012 14:16:53 +0000
Subject: [Pombelist] PhD position at the University of St Andrews for UK/EU
	student
Message-ID: <CB6BFACA.A067%sam31@st-andrews.ac.uk>

Dear All

Please forward the details of the opportunity below to anyone you think might be interested - a PDF poster for display is also attached.

----------------------------------

Fission yeast TFIIH: at the nexus of transcription and DNA repair

A PhD position is available at the University of St Andrews to study the biology of TFIIH complex in fission yeast. In this PhD project, available to any individual from the EU with a good first degree in the life sciences, you will build on recent progress in the reconstitution of TFIIH from fission yeast. Biochemical, genetic and structural studies of the complex and individual sub-complexes will be used to define their functions, mechanisms and interactions in DNA repair and transcription. For more information,  contact either of the project co-supervisors: Dr Stuart MacNeill (sam31 at st-and.ac.uk) or Prof Malcolm White (mfw2 at st-and.ac.uk).

----------------------------------

Many thanks and best wishes,

Stuart.

Dr Stuart A. MacNeill
Reader in Translational Biology
Biomedical Sciences Research Complex B306
University of St Andrews
North Haugh
St Andrews
FIFE KY16 9ST
UK

T: +44 1334 467 268
F: +44 1334 462 595
E: stuart.macneill at st-andrews.ac.uk
W: http://biology.st-andrews.ac.uk/macneill
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120223/66dbebe7/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: TFIIH_PhD_2012.pdf
Type: application/pdf
Size: 394313 bytes
Desc: TFIIH_PhD_2012.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120223/66dbebe7/attachment.pdf>

From t.sideri at ucl.ac.uk  Tue Feb 28 17:22:58 2012
From: t.sideri at ucl.ac.uk (Sideri, Dora)
Date: Tue, 28 Feb 2012 17:22:58 +0000
Subject: [Pombelist] cell viability assay using FACS
Message-ID: <D965734944251044BC1FF97A820C440307C35C80@AMSPRD0104MB099.eurprd01.prod.exchangelabs.com>

Hello everyone,



I would like to assay cell viability using flow cytometry. I was wondering if anyone have some experience with this and could send me a protocol.



Many Thanks

Dora



--------------------------------------------------
Dora Sideri
Department of Genetics, Evolution and Environment
and UCL Cancer Institute
University College London
Darwin Building, Gower Street,
WC1E 6BT, London,UK
--------
 P: +44 (0)20 3108 1606
 www.bahlerlab.info<http://www.bahlerlab.info>
--------------------------------------------------
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120228/9cb92688/attachment-0002.htm>

From luis.rokeach at umontreal.ca  Tue Feb 28 17:47:43 2012
From: luis.rokeach at umontreal.ca (Rokeach Luis)
Date: Tue, 28 Feb 2012 12:47:43 -0500
Subject: [Pombelist] cell viability assay using FACS
In-Reply-To: <D965734944251044BC1FF97A820C440307C35C80@AMSPRD0104MB099.eurprd01.prod.exchangelabs.com>
Message-ID: <CB727D6F.15677%luis.rokeach@umontreal.ca>

Hello Dora,

For reference, you can see the following of our publications.


Best,

Luis


 
Luis A. Rokeach, PhD
Professor
Department of Biochemistry
Universit? de Montr?al
C.P. 6128, succ. Centre-ville
Montr?al, Qc H3C 3J7 Canada
Phone 1(514) 343-6324; Fax 1(514) 343-2210
luis.rokeach at umontreal.ca
http://www.mapageweb.umontreal.ca/rokeach




Calnexin regulates apoptosis induced by inositol starvation in fission
yeast.
Gu?rin R, Beauregard PB, Leroux A, Rokeach LA.
PLoS One. 2009 Jul 16;4(7):e6244.

Pro-aging effects of glucose signaling through a G protein-coupled glucose
receptor in fission yeast.
Roux AE, Leroux A, Alaamery MA, Hoffman CS, Chartrand P, Ferbeyre G, Rokeach
LA.
PLoS Genet. 2009 Mar;5(3):e1000408. Epub 2009 Mar 6.

Calnexin is involved in apoptosis induced by endoplasmic reticulum stress in
the fission yeast.
Gu?rin R, Arseneault G, Dumont S, Rokeach LA.
Mol Biol Cell. 2008 Oct;19(10):4404-20. Epub 2008 Aug 13.


Regulation of chronological aging in Schizosaccharomyces pombe by the
protein kinases Pka1 and Sck2.
Roux AE, Quissac A, Chartrand P, Ferbeyre G, Rokeach LA.
Aging Cell. 2006 Aug;5(4):345-57. Epub 2006 Jul 5.





Le 12-02-28 12:22, ??Sideri, Dora?? <t.sideri at ucl.ac.uk> a ?crit?:

> Hello everyone,
> 
>  
> 
> I would like to assay cell viability using flow cytometry. I was wondering if
> anyone have some experience with this and could send me a protocol.
> 
>  
> 
> Many Thanks
> 
> Dora
> 
>  
> 
> --------------------------------------------------
> Dora Sideri
> Department of Genetics, Evolution and Environment
> and UCL Cancer Institute
> University College London
> Darwin Building, Gower Street,
> WC1E 6BT, London,UK
> --------
>  P: +44 (0)20 3108 1606
>  www.bahlerlab.info <http://www.bahlerlab.info>
> --------------------------------------------------
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120228/539e4ec9/attachment-0002.htm>

From clara_reis2002 at yahoo.co.uk  Tue Feb 28 19:16:25 2012
From: clara_reis2002 at yahoo.co.uk (Clara Reis)
Date: Tue, 28 Feb 2012 19:16:25 +0000 (GMT)
Subject: [Pombelist] pESP-1 or pESP-3 expression vectors
In-Reply-To: <CB727D6F.15677%luis.rokeach@umontreal.ca>
References: <D965734944251044BC1FF97A820C440307C35C80@AMSPRD0104MB099.eurprd01.prod.exchangelabs.com>
	<CB727D6F.15677%luis.rokeach@umontreal.ca>
Message-ID: <1330456585.9645.YahooMailNeo@web28602.mail.ukl.yahoo.com>

Hello everyone!

I am searching for pESP-1 vector ( Lu et al, 1997 Gene) or its derived pESP-3 (Stratagene). Unfortunately I don't find them commercially available.?
If anyone has them and is willing to send me a little I will be very grateful,?
Cheers!,
Clara

?
Clara C. Reis, PhD 
Telomere and Genome Stability Lab
Instituto Gulbenkian de Ci?ncia
Rua da Quinta Grande, 6
2780-156 Oeiras
Portugal
http://sites.igc.gulbenkian.pt/telomere/tgs/Welcome.html
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120228/c7f074e7/attachment-0002.htm>

From pluskal at oist.jp  Fri Mar 16 05:10:55 2012
From: pluskal at oist.jp (Tomas Pluskal)
Date: Fri, 16 Mar 2012 05:10:55 +0000
Subject: [Pombelist] Phthalate in EMM2
Message-ID: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>

Dear pombe community,

I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.

The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.

In particular, I would like to know what is the role of the phthalate in the medium.

Best regards,

Tomas



[cid:2CE68A67-01E1-4823-BAEB-10D615E5F7C4 at oist.jp]


[cid:5E20FCD6-CBC0-40E1-9453-04D64C655346 at oist.jp]


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/d98c7be9/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: EMM2 Mitchison.png
Type: image/png
Size: 128727 bytes
Desc: EMM2 Mitchison.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/d98c7be9/attachment.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: EMM2 Nurse.png
Type: image/png
Size: 254340 bytes
Desc: EMM2 Nurse.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/d98c7be9/attachment-0001.png>

From Ken.Sawin at ed.ac.uk  Fri Mar 16 08:16:50 2012
From: Ken.Sawin at ed.ac.uk (Ken Sawin)
Date: Fri, 16 Mar 2012 08:16:50 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
Message-ID: <9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>

Dear Tomas,

I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!

Best wishes,

Ken


On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:

> Dear pombe community,
> 
> I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.
> 
> The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.
> 
> In particular, I would like to know what is the role of the phthalate in the medium.
> 
> Best regards,
> 
> Tomas 
> 
> 
> 
> <EMM2 Mitchison.png>
> 
> 
> <EMM2 Nurse.png>
> 
> 
> ===============================================
> Tomas Pluskal
> G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
> 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
> TEL:  +81-98-966-8684
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk






-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.




From paul.nurse at royalsociety.org  Fri Mar 16 08:44:59 2012
From: paul.nurse at royalsociety.org (Nurse, Paul)
Date: Fri, 16 Mar 2012 08:44:59 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
	<9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
Message-ID: <1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>

Ken you are exactly right. Paul N
On 16 Mar 2012, at 08:16, Ken Sawin wrote:

> Dear Tomas,
> 
> I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!
> 
> Best wishes,
> 
> Ken
> 
> 
> On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:
> 
>> Dear pombe community,
>> 
>> I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.
>> 
>> The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.
>> 
>> In particular, I would like to know what is the role of the phthalate in the medium.
>> 
>> Best regards,
>> 
>> Tomas 
>> 
>> 
>> 
>> <EMM2 Mitchison.png>
>> 
>> 
>> <EMM2 Nurse.png>
>> 
>> 
>> ===============================================
>> Tomas Pluskal
>> G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
>> 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
>> TEL:  +81-98-966-8684
>> 
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://lists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> Kenneth E. Sawin, Ph.D.
> The Wellcome Trust Centre for Cell Biology
> School of Biological Sciences, University of Edinburgh
> Swann Building, Mayfield Road
> Edinburgh EH9  3JR
> United Kingdom
> 
> tel: 44-131-650-7064
> fax: 44-131-650-7360
> email: ken.sawin at ed.ac.uk
> 
> 
> 
> 
> 
> 
> -- 
> The University of Edinburgh is a charitable body, registered in
> Scotland, with registration number SC005336.
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom. 

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org

Registered charity no. 207043



From pluskal at oist.jp  Fri Mar 16 08:54:37 2012
From: pluskal at oist.jp (Tomas Pluskal)
Date: Fri, 16 Mar 2012 08:54:37 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
	<9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
	<1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>
Message-ID: <8398F6FE-EB90-435A-8149-1CEB51D4893C@oist.jp>

Dear Ken & Paul,

Thank you for the answer!

I suppose the reason for including the acetate or phthalate is to maintain the pH, is that correct?


Tomas


On Mar 16, 2012, at 5:44 PM, Nurse, Paul wrote:

Ken you are exactly right. Paul N
On 16 Mar 2012, at 08:16, Ken Sawin wrote:

Dear Tomas,

I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!

Best wishes,

Ken


On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:

Dear pombe community,

I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.

The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.

In particular, I would like to know what is the role of the phthalate in the medium.

Best regards,

Tomas



<EMM2 Mitchison.png>


<EMM2 Nurse.png>


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk<mailto:ken.sawin at ed.ac.uk>






--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom.

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org<mailto:ithelpdesk at royalsociety.org>

Registered charity no. 207043

===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120316/fc15436a/attachment-0002.htm>

From paul.nurse at royalsociety.org  Fri Mar 16 09:05:59 2012
From: paul.nurse at royalsociety.org (Nurse, Paul)
Date: Fri, 16 Mar 2012 09:05:59 +0000
Subject: [Pombelist] Phthalate in EMM2
In-Reply-To: <8398F6FE-EB90-435A-8149-1CEB51D4893C@oist.jp>
References: <31DB33BD-2A16-45B1-BC9D-4618A3B21575@oist.jp>
	<9857A31F-56D4-4EDA-AEF8-2B4AF71D97E9@ed.ac.uk>
	<1350224A-22A2-46E7-B92B-C2CB72A3D8F7@royalsociety.org>
	<8398F6FE-EB90-435A-8149-1CEB51D4893C@oist.jp>
Message-ID: <0DF7D88A-6341-440E-A1AE-5FE39CA5F53F@royalsociety.org>

Yes P
On 16 Mar 2012, at 08:54, Tomas Pluskal wrote:

Dear Ken & Paul,

Thank you for the answer!

I suppose the reason for including the acetate or phthalate is to maintain the pH, is that correct?


Tomas


On Mar 16, 2012, at 5:44 PM, Nurse, Paul wrote:

Ken you are exactly right. Paul N
On 16 Mar 2012, at 08:16, Ken Sawin wrote:

Dear Tomas,

I think phthalate was substituted for acetate in order to reduce clumping of cells in Paul's screen and/or analysis of cdc and wee mutants.  Someone a little older than me may remember the details!

Best wishes,

Ken


On 16 Mar 2012, at 05:10, Tomas Pluskal wrote:

Dear pombe community,

I was wondering if anyone knows the reason why Paul Nurse modified the EMM2 medium.

The original EMM2 by J.M. Mitchison (1970) contained sodium acetate and sodium dihydrogen orthophosphate, which were replaced by P. Nurse (1975) with potassium hydrogen phthalate and disodium hydrogen orthophosphate. However, P. Nurse's paper did not mention the motivation behind this change.

In particular, I would like to know what is the role of the phthalate in the medium.

Best regards,

Tomas



<EMM2 Mitchison.png>


<EMM2 Nurse.png>


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk<mailto:ken.sawin at ed.ac.uk>






--
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.


_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom.

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org<mailto:ithelpdesk at royalsociety.org>

Registered charity no. 207043

===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
TEL:  +81-98-966-8684

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://lists.sanger.ac.uk/mailman/listinfo/pombelist

*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom. 

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org

Registered charity no. 207043



From benoit.arcangioli at pasteur.fr  Mon Mar 12 09:57:10 2012
From: benoit.arcangioli at pasteur.fr (Benoit Arcangioli)
Date: Mon, 12 Mar 2012 10:57:10 +0100
Subject: [Pombelist] EMBO Course on Fission yeast 1-13th July
Message-ID: <4F5DC876.1060603@pasteur.fr>

Dear all,
I'm please to inform you that the EMBO course on fission Yeast is open 
for registration:
http://events.embo.org/12-fission-yeast
Cheers
Iain Hagan
Olaf Nielsen
Benoit Arcangioli
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Poster embo-fission yeast.psd
Type: application/x-photoshop
Size: 7929903 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120312/93d05597/attachment.bin>

From geetanjali.sundaram at gmail.com  Wed Mar 21 11:09:25 2012
From: geetanjali.sundaram at gmail.com (Geetanjali Sundaram)
Date: Wed, 21 Mar 2012 06:09:25 -0500
Subject: [Pombelist] EMM and G418
Message-ID: <CAMKSyE4Eyf0+QXiLAW9gcUg7Ug=fEX2R096Y2UC3KiF2Ub9Yuw@mail.gmail.com>

Dear pombelist members,
can anyone tell me whether G418 works well in EMM-Ura media. i have
been trying to make a mutant for which i do the selection on this
media. but when i reinoculate the cells selected on EMM-Ura+G418 into
YES+G418, they dont grow,while they do grow on EMM-Ura media.
Regards
Geetanjali
-- 
Geetanjali Sundaram
Assistant Professor
Department of Biochemistry
Ballygunje Science College
University of Calcutta
35 Ballygunje Circular Road
Kolkata-700019
Ph-9433191982



From esmaakkaya at yahoo.com  Wed Mar 21 11:53:13 2012
From: esmaakkaya at yahoo.com (Esma AKKAYA)
Date: Wed, 21 Mar 2012 04:53:13 -0700 (PDT)
Subject: [Pombelist] EMM and G418
In-Reply-To: <CAMKSyE4Eyf0+QXiLAW9gcUg7Ug=fEX2R096Y2UC3KiF2Ub9Yuw@mail.gmail.com>
References: <CAMKSyE4Eyf0+QXiLAW9gcUg7Ug=fEX2R096Y2UC3KiF2Ub9Yuw@mail.gmail.com>
Message-ID: <1332330793.34764.YahooMailNeo@web112517.mail.gq1.yahoo.com>

Dear Dr Sundaram,
?
I recently tried EMM+G418 to make sure if it works or not. I compared my strains on EMM only and EMM+G418.?Unfortunately G418?didn't work at all on EMM for my strains. The reason?may be?that the amonium chloride in EMM media is competing with the drug so you need to substitute amonium with something else. This is also well explained in Dr. Forsburg's PombeNet page (http://www-bcf.usc.edu/~forsburg/drugs.html). PMG media is suggested instead of EMM for G418.
?
PMG recipe: http://www-bcf.usc.edu/~forsburg/media.html#PMG
?
I haven't tried yet but in your case you may have a problem with ura- strains?because it is also added as a bonus on PombeNet page where they say Ura- cells grow more similarly to Ura+ cells on PMG. It may work if you plate your cells first on YES+G418 and then replicate them on EMM-ura. 
?
Best Regards,
?
Esma Ucisik-Akkaya
?
?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Esma Ucisik-Akkaya
Grad student?- NEIMAN LAB
Graduate Program in Molecular Genetics & Microbiology
Stony Brook University
323?Life Sciences Building
Stony Brook, NY 11794-5222
?
Lab phone: 1-(631)-632-1421
Lab page: http://www.stonybrook.edu/biochem/neiman/lab.html


________________________________
From: Geetanjali Sundaram <geetanjali.sundaram at gmail.com>
To: pombelist at sanger.ac.uk 
Sent: Wednesday, March 21, 2012 7:09 AM
Subject: [Pombelist] EMM and G418

Dear pombelist members,
can anyone tell me whether G418 works well in EMM-Ura media. i have
been trying to make a mutant for which i do the selection on this
media. but when i reinoculate the cells selected on EMM-Ura+G418 into
YES+G418, they dont grow,while they do grow on EMM-Ura media.
Regards
Geetanjali
-- 
Geetanjali Sundaram
Assistant Professor
Department of Biochemistry
Ballygunje Science College
University of Calcutta
35 Ballygunje Circular Road
Kolkata-700019
Ph-9433191982

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://lists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120321/f9d4ff4d/attachment-0002.htm>

From daniel.scott at path.ox.ac.uk  Fri Mar 30 17:02:14 2012
From: daniel.scott at path.ox.ac.uk (Daniel Scott)
Date: Fri, 30 Mar 2012 17:02:14 +0100
Subject: [Pombelist] ade6 transcription start site
In-Reply-To: <4F13F992.2010408@sanger.ac.uk>
References: <44045.125.19.68.205.1326534802.squirrel@webmail.imtech.res.in>
	<4F13F992.2010408@sanger.ac.uk>
Message-ID: <CANbSe_ky3qu4prBT+QYcOByX+zgNLbDXS6hL8T6FTY_WOhKQPA@mail.gmail.com>

Hi there fellow pombologists,

A quick query regarding auxotrophic supplements to minimal media - we've
been looking at using adenine sulphate rather than the standard adenine
hydrochloride and wonder if anyone (a) has any experience of this and (b)
if there's any reason why it may not work.  The standard salt solution
added to minimal media does contain Na2SO4 at 14.1mM final concentration
which would only increase to ~15mM, so shouldn't be a huge change in the
anion concentration.  Looking forward to hearing of your sage insights!

Regards,

Dan Scott
Norbury Lab
Sir William Dunn School of Pathology
University of Oxford
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120330/5f92012b/attachment-0002.htm>

From ozan.aygun at nih.gov  Tue Apr  3 03:04:19 2012
From: ozan.aygun at nih.gov (Aygun, Ozan (NIH/NCI) [F])
Date: Mon, 2 Apr 2012 22:04:19 -0400
Subject: [Pombelist] Comprehensive promoter resource for S.pombe
Message-ID: <CB9FD2E3.2102%ayguno@mail.nih.gov>

Dear Pombe Investigators,

I am wondering if any of you can recommend me a research article data set or a website directory that compiled a comprehensive list of defined S.pombe promoter sequences? There is a database for S.cerevisiae promoters, but I was unable to locate something analogous for fission yeast.

Sincerely,

Ozan Aygun


**************************************
Ozan Aygun, Ph.D.

National Cancer Institute
National Institutes of Health
Bethesda, Maryland 20892-4260
USA

Tel: 301-594-5607
E-mail: ozan.aygun at nih.gov
**************************************





From mah79 at cam.ac.uk  Tue Apr  3 14:09:02 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Tue, 3 Apr 2012 14:09:02 +0100 (BST)
Subject: [Pombelist] Comprehensive promoter resource for S.pombe
In-Reply-To: <CB9FD2E3.2102%ayguno@mail.nih.gov>
References: <CB9FD2E3.2102%ayguno@mail.nih.gov>
Message-ID: <Pine.LNX.4.64.1204031403350.10563@hermes-2.csi.cam.ac.uk>

Dear Dr. Ozan Aygun,

We do not have promoter sequences annotated in PomBase (nor its 
predecessor GeneDB), and I don't know of a database for S. pombe 
equivalent to the S. cerevisiae SCPD.

The closest thing we have at present is a curated list of DNA binding 
consensus sequences, most of which are promoter elements such as 
transcription factor binding sites; we have recently made these available 
on a web page:

http://www.pombase.org/dna-binding-sites

I hope this page is of some use even though it's not exactly what you're 
asking for. We would be very happy to host or link to promoter data for 
pombe if anyone in the community can make such data available, or knows of 
a relevant resource. We also welcome specific additions or corrections to 
the DNA binding site page.

Best regards,
Midori

Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk


On Mon, 2 Apr 2012, Aygun, Ozan (NIH/NCI) [F] wrote:

> Dear Pombe Investigators,
>
> I am wondering if any of you can recommend me a research article data 
> set or a website directory that compiled a comprehensive list of defined 
> S.pombe promoter sequences? There is a database for S.cerevisiae 
> promoters, but I was unable to locate something analogous for fission 
> yeast.
>
> Sincerely,
>
> Ozan Aygun
>
>
> **************************************
> Ozan Aygun, Ph.D.
>
> National Cancer Institute
> National Institutes of Health
> Bethesda, Maryland 20892-4260
> USA
>
> Tel: 301-594-5607
> E-mail: ozan.aygun at nih.gov
> **************************************
>
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://lists.sanger.ac.uk/mailman/listinfo/pombelist
>



From j.bahler at ucl.ac.uk  Mon Apr 16 19:52:01 2012
From: j.bahler at ucl.ac.uk (Bahler, Jurg)
Date: Mon, 16 Apr 2012 18:52:01 +0000
Subject: [Pombelist] Tool to predict protein-protein interactions
Message-ID: <74249C6FD1C7CB4CB0E1451F04F8D194E045BA@DB3PRD0104MB132.eurprd01.prod.exchangelabs.com>

PInt (Pombe Interactome) is a tool that allows you to retrieve predictions of S. pombe protein interactions, based on more than
100 features. The predictions were generated using two machine learning algorithms, and the confidence scores returned are used to rank interactions according to their probability.
PInt can be used for hypothesis generation and for exploring the interactome neighbourhoods of proteins of interest, which can be visualized through a user-friendly interface: http://www.bahlerlab.info/PInt/

For details see:
Pancaldi V, Sara? ?., Rallis C., McLean J.R., P?evorovsk? M., Gould K., Beyer A. and B?hler J. (2012). Predicting the fission yeast protein interaction network.
G3, 2(4), 453-467 (http://www.g3journal.org/content/2/4/453.abstract)

Enjoy,
-J?rg

---
J?rg B?hler
Professor of Systems Biology

University College London
Department of Genetics, Evolution & Environment
and UCL Cancer Institute
Darwin Building, Gower Street
London WC1E 6BT
U.K.

P: +44(0)20 3108 1602 (Internal x51602)
F: +44(0)20 7679 7096
E: j.bahler at ucl.ac.uk<mailto:j.bahler at ucl.ac.uk>
W: www.bahlerlab.info<http://www.sanger.ac.uk/PostGenomics/S_pombe/>

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120416/9e2cece4/attachment-0002.htm>

From mah79 at cam.ac.uk  Tue Apr 17 16:00:48 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Tue, 17 Apr 2012 16:00:48 +0100 (BST)
Subject: [Pombelist] PomBase sequence download
Message-ID: <Pine.LNX.4.64.1204171428520.16854@hermes-2.csi.cam.ac.uk>

Dear Pombe Researchers,

We are pleased to announce that sequence downloads are now working 
properly from gene pages at www.pombase.org. We have also ensured that 
exon coordinates are listed in the correct order, and added CDS 
coordinates to the summary at the top of each gene page.

Many thanks to all of our users, especially those who have reported the 
issues that we have recently addressed. Please let us know if you have any 
problems with the new or fixed features, or if you have any other comments 
about PomBase.

Best regards,
Midori


Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk




From oliver at singerinstruments.com  Thu Apr 26 10:20:37 2012
From: oliver at singerinstruments.com (Ollie Thompson)
Date: Thu, 26 Apr 2012 09:20:37 +0000
Subject: [Pombelist] Scientific Advisor role with Singer Instruments (UK)
Message-ID: <C59C476009C0BB46840014103F60535737BBE55A@SINCOSERVER2.SINCOINTL.local>

Dear Pombe list members,



Position available: Scientific Advisor (South-West UK)



We are seeking a new member of the Singer Instruments team!



We're looking for an outgoing, motivated, and enthusiastic scientist with an interest in biotechnology hardware and applications, who wishes to keep in touch with laboratory life.



A strong background in science is required, ideally with post-graduate experience in yeast genetics, microbiology, or cell biology. Prior consultancy experience is not necessary as training will be given. What is required is a pleasant, friendly, outgoing personality...table-football experience is desirable!



The position involves a variety of day-to-day roles mixing science, marketing, R&D, sales, and accounts management, and includes:



* promoting and raising awareness of our existing products to new and existing customers
* liaising with existing and potential customers to understand their research needs
* participating in smart marketing strategies and campaigns
* seeking new areas of application for our instruments within industrial and academic research
* managing customer information and sales data
* advising on the strategic direction for new scientific devices and contributing to their design



The ideal candidate will have an interest in acquiring knowledge and skills in a commercial environment, as well as maintaining an awareness of scientific developments. There is a healthy travel budget, and the position will involve some travel to domestic and international scientific meetings to engage with active research scientists.



This position would suit someone who is seeking to apply their scientific expertise to practical biotechnology applications, whilst gaining business skills and experience.


Apply at http://bit.ly/SingerJobs

Dr Oliver Thompson | Scientific Advisor
SingerInstrument Co. Ltd.
A responsibility to science!
www.singerinstruments.co.uk<blocked::http://www.singerinstruments.co.uk/>
yeast at singerinst.co.uk<blocked::mailto:yeast at singerinst.co.uk>
T: +44 (0)1984 640226
F: +44 (0)1984 641166
M: +44 (0) 7972323822

Awesome: Sign up to our newsletter!<http://www.singerinst.co.uk/test/index.php?option=com_content&task=view&id=208&Itemid=884>

Boring: Read our smallprint<http://www.singerinst.co.uk/test/index.php?option=com_content&task=view&id=212>

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120426/96348e7f/attachment-0002.htm>

From matthew.oconnell at mssm.edu  Tue May 15 01:03:47 2012
From: matthew.oconnell at mssm.edu (Oconnell, Matthew)
Date: Tue, 15 May 2012 00:03:47 +0000
Subject: [Pombelist] Postdoc positions
Message-ID: <5001ED0A-936D-456C-9E91-910F6181486D@mssm.edu>

Hello everyone

I have several postdoc positions opening in my lab to study various aspects of genome integrity in pombe.

Please alert anyone you know who is looking for a position to email me, and I can give more details.

Many thanks

Matthew

__________________________________
Matthew J. O?Connell, Ph.D.
Mount Sinai School of Medicine
Department of Oncological Sciences
One Gustave L. Levy Place - Box 1130
New York, New York  10029
Phone:  (212) 659-5468
Fax:      (212) 987-2240
matthew.oconnell at mssm.edu<mailto:matthew.oconnell at mssm.edu>

Express Mail/FedEx:
1425 Madison Ave - Room 15-70
New York, New York  10029


-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120515/8442fb61/attachment.htm>

From poonam at imtech.res.in  Tue May 15 12:38:09 2012
From: poonam at imtech.res.in (Poonam Shukla)
Date: Tue, 15 May 2012 17:08:09 +0530 (IST)
Subject: [Pombelist] (no subject)
Message-ID: <48035.125.19.68.203.1337081889.squirrel@webmail.imtech.res.in>

Dear pombe researchers,
I have a query regarding some technical problem that I am facing these days.
Actually we have tagged our gene of interest with GFP using Bahler?s method with
G418 selection. The tag tends to be lost when culture is grown mitotically or
during meiosis if crossed with another untagged strain in absence of the drug.
Surprisingly even when crossed with another tagged strain in presence of G418 no
viable spores appear on G418 plates during tetrad dissection.
Can anyone suggest us a solution?

Thanks,
Poonam

______________________________________________________________________
?????????? ???????????? ??????? (????????? ???????? ???????? ?????)
Institute of Microbial Technology (A CONSTITUENT ESTABLISHMENT OF CSIR)
?????? 39 ?, ???????? / Sector 39-A, Chandigarh
??? ???/PIN CODE :160036
??????/EPABX :0172 6665 201-202


From J.Davey at warwick.ac.uk  Sat May 19 16:46:57 2012
From: J.Davey at warwick.ac.uk (Davey, John)
Date: Sat, 19 May 2012 15:46:57 +0000
Subject: [Pombelist] Group Leader posts at Warwick Medical School
Message-ID: <364019EB33212C46BF9D1175A8F5BE2202FAF0@DB3PRD0104MB144.eurprd01.prod.exchangelabs.com>

Dear Pombelist member

The Division of Biomedical Cell Biology at Warwick Medical School is looking to recruit Group Leaders (at Professor and Associate Professor levels) to carry out research that addresses a medically relevant cell biological problem.   The Division offers a supportive environment for biochemical, genetical and biophysical research across a range of model systems.

For information about the current members of the Division, and their research interests:
See http://www2.warwick.ac.uk/fac/med/research/biomedical
*       Robert Cross - Mechanochemistry of kinesin and tubulin
*       Jacob Dalgaard - DNA replication
*       John Davey - Intracellular signalling through G proteins
*       Graham Ladds - Modelling spatiotemporal control of cell signalling
*       Andrew McAinsh - Mechanics of chromosome separation
*       Jonathan Millar - Cell cycle control mechanisms
*       Masanori Mishima - Mechanics and control of cytokinesis
*       Anne Straube - Cytoskeletal organization and cell migration

For further details about the posts:
Professor - https<https://secure.admin.warwick.ac.uk/webjobs/jobs/academic/job12687.html>://secure.admin.warwick.ac.uk/webjobs/jobs/academic/job12687.html<https://secure.admin.warwick.ac.uk/webjobs/jobs/academic/job12687.html>
Associate Professor - https://secure.admin.warwick.ac.uk/webjobs/jobs/academic/job10053.html

Interested candidates are encouraged to contact John Davey (Head of Division):
J.Davey at Warwick.ac.uk<mailto:J.Davey at Warwick.ac.uk> 02476 524204

Regards, John
--
John Davey, PhD DSc
Head of Division, Division of Biomedical Cell Biology
Warwick Medical School, The University of Warwick, Coventry, CV4 7AL
Tel: +44 (0) 24 76524204
J.Davey at warwick.ac.uk<mailto:J.Davey at warwick.ac.uk>
http://www2.warwick.ac.uk/fac/med/research/biomedical/

Divisional Secretary: Gemma Wild
Tel: +44 (0) 24 76151154
G.Wild at Warwick.ac.uk<mailto:G.Wild at Warwick.ac.uk>

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120519/a6eacbc7/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Group Leader posts at Warwick.pdf
Type: application/pdf
Size: 347024 bytes
Desc: Group Leader posts at Warwick.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120519/a6eacbc7/attachment-0001.pdf>

From pei-yun.wu at univ-rennes1.fr  Sun May 20 15:47:46 2012
From: pei-yun.wu at univ-rennes1.fr (Jenny Wu)
Date: Sun, 20 May 2012 16:47:46 +0200
Subject: [Pombelist] Ph. D. position
Message-ID: <FBDCA1D4-BD07-477A-8ACD-87EBD972B15E@univ-rennes1.fr>

Dear all,

A fully-funded Ph. D. position is available starting Fall, 2012 in the Genome Duplication and Maintenance group of Dr. Pei-Yun Jenny Wu at the Institute of Genetics and Development of Rennes (IGDR) in Brittany, France. Our team uses the fission yeast to investigate the control of DNA replication and the role of the multiple layers regulating DNA synthesis in the maintenance of genome integrity. We are seeking candidates to conduct a project that focuses on the fundamental parameters that determine the organization and selection of origins of DNA replication.

For further information regarding the position and the institute, please see the attached PDF file as well as the following website: 

http://igdr.univ-rennes1.fr/english/


Please contact me if you need any additional information. Selected candidates will then need to apply to the Doctoral School of the University of Rennes by June 11, 2012.

Thank very much you for your time.


Sincerely,

Jenny Wu


**************************************************************************
Laboratory of Genome Duplication and Maintenance
Institute of Genetics and Development of Rennes, CNRS UMR 6290
2 avenue du Professeur L?on Bernard
Rennes, France 35043
email: pei-yun.wu at univ-rennes1.fr
phone: +33 2 23 23 44 06 



-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120520/cfed5cbb/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: PhD_ad.pdf
Type: application/pdf
Size: 826456 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120520/cfed5cbb/attachment-0001.pdf>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120520/cfed5cbb/attachment-0003.htm>

From jblyth1 at staffmail.ed.ac.uk  Thu May 31 10:16:35 2012
From: jblyth1 at staffmail.ed.ac.uk (BLYTH Julie)
Date: Thu, 31 May 2012 10:16:35 +0100
Subject: [Pombelist] protoplast fusion method
Message-ID: <FE652A7F-D079-4040-A1B2-5649F970CD20@staffmail.ed.ac.uk>

Dear all

We are wanting to make diploids by the protoplast fusion method.  The Novozyme enzyme is no longer available and I was wondering what people are using instead?

Thanks in advance for your help.

Julie Blyth


Wellcome Trust Centre for Cell Biology
Michael Swann Building, The King's Buildings
University of Edinburgh, Mayfield Road
Edinburgh 
EH9 3JR
Scotland, UK 
-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.



From Sarah.Lambert at curie.fr  Thu May 31 16:56:08 2012
From: Sarah.Lambert at curie.fr (Lambert Sarah)
Date: Thu, 31 May 2012 15:56:08 +0000
Subject: [Pombelist] protoplast fusion method
In-Reply-To: <FE652A7F-D079-4040-A1B2-5649F970CD20@staffmail.ed.ac.uk>
References: <FE652A7F-D079-4040-A1B2-5649F970CD20@staffmail.ed.ac.uk>
Message-ID: <B68D2365E185A6469F4DCD245E9D5BF10844936F@mbxparis01.recherche.curie.fr>

Dear all
We used the protocol protoplast fusion 1 from the Fission Yeast Handbook.
We replaced the novozyme by 12 mg of lysing enzyme/ml (Sigma L1412) and 10mg/ml of zymolyase 20T in KCl 0.65M.
We plated cells on selective media, 1M sorbitol, 1% agar.
Hope it works for you.
All the best.
Sarah


Sarah Lambert
Research Scientist/CR1
Institut Curie/CNRS
UMR3348
Bat 110
Centre universitaire Paris-Sud XI
Orsay 91405
France
phone: 00 33 1 69 86 71 91
Fax: 00 33 1 69 86 94 29

http://www.curie.fr/equipe/366
http://www.curie.fr/equipe/366/lang/_gb

-----Message d'origine-----
De?: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] De la part de BLYTH Julie
Envoy??: jeudi 31 mai 2012 11:17
??: pombelist at sanger.ac.uk
Objet?: [Pombelist] protoplast fusion method

Dear all

We are wanting to make diploids by the protoplast fusion method.  The Novozyme enzyme is no longer available and I was wondering what people are using instead?

Thanks in advance for your help.

Julie Blyth


Wellcome Trust Centre for Cell Biology
Michael Swann Building, The King's Buildings University of Edinburgh, Mayfield Road Edinburgh
EH9 3JR
Scotland, UK
--
The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.


_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://publists.sanger.ac.uk/mailman/listinfo/pombelist


From colm.ryan at ucd.ie  Mon Jun 11 19:00:15 2012
From: colm.ryan at ucd.ie (Colm Ryan)
Date: Mon, 11 Jun 2012 19:00:15 +0100
Subject: [Pombelist] New S. pombe Genetic Interactome
Message-ID: <CAH+PQBXa10m=2+8tUNcq6weJ-t3J1XRtyhX4vFgSK5vZGNH2Kg@mail.gmail.com>

We are pleased to announce the availability of the largest genetic
interaction map in S. pombe to date. This map contains ~1.6 million
measured interactions and covers more than 60% of the non-essential genes
from virtually all major biological processes.  We trust it will be a
valuable resource for the S. pombe community.

To make our data easily accessible to other researchers, we have created a
supporting website at http://interactome-cmp.ucsf.edu/pombe2012/modules/

In addition to tools for searching for individual interactions, this site
contains lists of functional modules identified from the data - groups of
genes which exhibit highly similar genetic interaction profiles, indicating
that they belong to common complexes or pathways. We foresee these
functional module definitions being extremely useful for the functional
annotation of uncharacterized genes, 320 of which are contained in our
dataset.

The associated paper "Hierarchical Modularity and the Evolution of Genetic
Interactomes across Species" is available in Molecular Cell (
http://www.cell.com/molecular-cell/fulltext/S1097-2765(12)00444-3).

Should you have any queries about the data, please contact colm.ryan at ucd.ie

Best,

Assen Roguev, Colm J. Ryan, Nevan J. Krogan
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120611/cc52d2b0/attachment.htm>

From steven.sanders at case.edu  Fri Jun 15 03:04:20 2012
From: steven.sanders at case.edu (Steven Sanders)
Date: Thu, 14 Jun 2012 19:04:20 -0700
Subject: [Pombelist] Lab closed
Message-ID: <72830B92-3C7E-4EC9-8D13-B84CE904A5C5@case.edu>

To the pombe community,

I would like to briefly announce that my research lab is now closed as I have moved on to the biotech world.  I would like to thank all of those individuals that I have had the pleasure to interact with over the past 10 years and I will genuinely miss the collegiality of the pombe community.

All strains and plasmids from past publications have been transferred to Kurt Runge's laboratory at the Cleveland Clinic.  Reagent requests can go directly to Kurt (rungeK at ccf.org) and the information below will get you to me for other questions.

Cheers,
Steve

___________________________
Steven L. Sanders, Ph.D.
Senior Research Scientist
Cibuss LLC
6455 Nancy Ridge Drive, Suite 100
San Diego, CA 92121
(858) 450-0008, ext. 109
ssanders at cibusllc.com





From hniki at nig.ac.jp  Fri Jun 15 03:44:31 2012
From: hniki at nig.ac.jp (NIKI Hironori)
Date: Fri, 15 Jun 2012 11:44:31 +0900
Subject: [Pombelist] New Bioresource for Sz. japonicus Research
Message-ID: <08EF789B-5A82-4C78-B591-8F1CD642C54F@nig.ac.jp>

Dear all,

We are pleased to announce that an insertion marker collection of Sz. japonicus (IMACS) is now open for Japonicus researchers.

The collection includes 29 strains that bear a positive-negative selection marker at different loci, 
and the marker is inserted every 500?kb in the genome of Sz. japonicus.

For more information and request, to JapoNet, http://night.nig.ac.jp/labs/MicroGen/japonet/

Best regards,
NIKI

NIKI, Hironori Ph.D  Professor
Microbial Genetics Laboratory
Genetic Strains Research Center
National Institute of Genetics
1111, Yata, Mishima, SHIZUOKA, 
JAPAN 411-8540
FAX: +8-55-981-6826
Tel: +8-55-981-6870





From jacqueline.hayles at cancer.org.uk  Thu Jun 21 16:07:24 2012
From: jacqueline.hayles at cancer.org.uk (Jacqueline Hayles)
Date: Thu, 21 Jun 2012 16:07:24 +0100
Subject: [Pombelist] Pombe Club
Message-ID: <CC08F73C.E162%jacqueline.hayles@cancer.org.uk>

Hi Everyone

I'm going to organise a Pombe club at the LRI on September 25th or near to that date, unless anyone would like to organise a meeting.  I would be grateful for offers to speak or suggestions for speakers .  I hope to hear from you soon.

All the best

Jacky

Jacqueline Hayles
Cell Cycle Laboratory
Cancer Research UK, London Research Institute
44 Lincoln's Inn Fields
London WC2A 3LY, UNITED KINGDOM

Tel ::: +44 (0) 20 7269 3105
Fax ::: +44 (0) 20 7269 3258
email   jacqueline.hayles at cancer.org.uk

[cid:C57ADCC3-47F7-4CD1-A278-EC39747E51FD]



NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered in England and Wales
Company Registered Number: 4325234.
Registered Charity Number: 1089464 and Scotland SC041666
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120621/d9f2070a/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 8E841165-810C-4AE2-9EEE-1E45A81D22C2[1].png
Type: image/png
Size: 61902 bytes
Desc: 8E841165-810C-4AE2-9EEE-1E45A81D22C2[1].png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120621/d9f2070a/attachment-0001.png>

From sandip.de at nih.gov  Sat Jun 23 18:21:54 2012
From: sandip.de at nih.gov (De, Sandip (NIH/NICHD) [F])
Date: Sat, 23 Jun 2012 13:21:54 -0400
Subject: [Pombelist] expression of mat locus
Message-ID: <7C75F8A72217D64DBD5914C4AEFEA9D61B3F560795@NIHMLBX15.nih.gov>

Hi can anyone tell me at which particular stage of cell cycle pombe mat1 locus is transcribed? I looked into the pombase for expression profiling of the locus in different cell cycle but no clear information is provided. Any information is much appreciated.

Thanks,
Sandip De
NICHD/NIH
Bethesda
US 20892

From jacqueline.hayles at cancer.org.uk  Mon Jun 25 12:40:15 2012
From: jacqueline.hayles at cancer.org.uk (Jacqueline Hayles)
Date: Mon, 25 Jun 2012 12:40:15 +0100
Subject: [Pombelist] Pombe Club
Message-ID: <CC0E0CAF.E1D0%jacqueline.hayles@cancer.org.uk>

Hi Everyone

Just to let you know we have finalised September 18th 2012 as the date for the next Pombe Club.  I will forward a programme soon.

Regards

Jacky

Jacqueline Hayles
Cell Cycle Laboratory
Cancer Research UK, London Research Institute
44 Lincoln's Inn Fields
London WC2A 3LY, UNITED KINGDOM

Tel ::: +44 (0) 20 7269 3105
Fax ::: +44 (0) 20 7269 3258
email   jacqueline.hayles at cancer.org.uk

[cid:F3A78AB8-118F-49A5-BBEA-89E2029F641A]




NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered in England and Wales
Company Registered Number: 4325234.
Registered Charity Number: 1089464 and Scotland SC041666
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120625/cccd5d8f/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 8E841165-810C-4AE2-9EEE-1E45A81D22C2.png
Type: image/png
Size: 61902 bytes
Desc: 8E841165-810C-4AE2-9EEE-1E45A81D22C2.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120625/cccd5d8f/attachment-0001.png>

From Ken.Sawin at ed.ac.uk  Thu Jun 28 16:25:19 2012
From: Ken.Sawin at ed.ac.uk (Ken Sawin)
Date: Thu, 28 Jun 2012 16:25:19 +0100
Subject: [Pombelist] Postdoc position available in Edinburgh
Message-ID: <924E7CD7-2AC5-4C26-87F1-DD9FD2C9282E@ed.ac.uk>

Dear All,

I have a postdoc position available in my lab--in this case, it's not a "typical" yeast genetics/cell biology project, but I would be grateful if you could post the attachment and/or pass it on to anyone you think may be interested.

Thanks,

Ken


-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.

-------------- next part --------------
A non-text attachment was scrubbed...
Name: Postdoc_MT_nucleation.pdf
Type: application/pdf
Size: 1454777 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120628/0ef3f089/attachment-0001.pdf>
-------------- next part --------------


 
Kenneth E. Sawin, Ph.D.
The Wellcome Trust Centre for Cell Biology
School of Biological Sciences, University of Edinburgh
Swann Building, Mayfield Road
Edinburgh EH9  3JR
United Kingdom

tel: 44-131-650-7064
fax: 44-131-650-7360
email: ken.sawin at ed.ac.uk






From damien.coudreuse at univ-rennes1.fr  Sun Jul  1 17:45:44 2012
From: damien.coudreuse at univ-rennes1.fr (Damien_IGDR)
Date: Sun, 1 Jul 2012 18:45:44 +0200
Subject: [Pombelist] Post-doc position available
Message-ID: <14211570-F2B1-433C-BF84-0B05C6BBE732@univ-rennes1.fr>

Dear all,

A post-doctoral position is available in my lab at the Institute of Genetics and Development of Rennes, France (http://igdr.univ-rennes1.fr/english/). My group will investigate different aspects of cell cycle progression in fission yeast, including its variability, dynamics and evolution. For further information, please see the attached PDF file.

Thank you very much.

Best wishes,

Damien Coudreuse

*************************
Damien Coudreuse
Institute of Genetics and Development
CNRS UMR 6061
2, Avenue du Pr. Leon Bernard
35043 Rennes Cedex
France
Phone: +33 (0)2 23234437

-------------- next part --------------
A non-text attachment was scrubbed...
Name: Postdoc-04:12.pdf
Type: application/pdf
Size: 386881 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120701/8a3ae9b3/attachment-0001.pdf>

From mah79 at cam.ac.uk  Mon Jul  2 12:08:14 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Mon, 2 Jul 2012 12:08:14 +0100 (BST)
Subject: [Pombelist] Major update to PomBase web site
Message-ID: <Pine.LNX.4.64.1206291632200.16822@hermes-2.csi.cam.ac.uk>

Dear pombe researchers,

We are pleased to announce that the PomBase web site, www.pombase.org, is 
now fully live; the preview phase has ended. The site has been updated 
with an assortment of new features, datatypes, and bug fixes.

More recent data, reflecting additions and changes through March 20, 2012, 
are now available on gene pages and in search results.

The updated site features a Gene List Search that provides behavior 
equivalent to GeneDB's List Download. You can now type or paste lists into 
the Gene Systematic IDs and Gene Names filters, and use the Query History 
to combine a gene list search with other search options. For convenience, 
there is a direct link to a search page pre-configured to accept a list of 
systematic IDs available in the Find menu, on the Find page, and here: 
http://www.pombase.org/spombe/query/builder?filter=12

  The Advanced Search also now offers:
  - options to search GO, FYPO, and other ontologies by term name or ID;
  - autocomplete for ontology term name search;
  - ability to search for genes in a region, such as centromeres or 
telomeres;
  - improved organization of filter selections.

We have also fixed a Sequence Download error reported by some users, so 
that the "CDS", CDS + UTRs", and "CDS + UTRs + Introns" options now 
retrieve the correct sequences.

In addition, numerous minor improvements have been made. Please send any 
questions or comments on the PomBase web site to us at 
<helpdesk at pombase.org>.

Best wishes,
The PomBase Team



From mah79 at cam.ac.uk  Tue Jul  3 11:23:22 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Tue, 3 Jul 2012 11:23:22 +0100 (BST)
Subject: [Pombelist] PomBase - flanking region sequence
Message-ID: <Pine.LNX.4.64.1207031118480.2194@hermes-2.csi.cam.ac.uk>

Dear pombe researchers,

We regret to inform you that, although one bug in the Sequence Download 
has indeed been fixed, as reported yesterday, another problem has cropped 
up. The custom download feature is not working properly, and does not 
retrieve flanking regions.

We will alert you as soon as this problem is corrected. In the meantime, 
we appreciate your patience.

Many thanks to Charlie Hoffman for noticing and reporting this problem. 
Please keep sending your comments, positive and negative, so that we can 
continue to improve PomBase.

Thank you,
Midori

---------------------------
Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk




From BarnesL at uthscsa.edu  Tue Jul  3 13:19:33 2012
From: BarnesL at uthscsa.edu (Barnes, Larry D)
Date: Tue, 3 Jul 2012 12:19:33 +0000
Subject: [Pombelist] unsubscribe
Message-ID: <4A3D62CDD3B70D4EA31E786B8CC6FC780E6776@EX-CCF-MBX2.win.uthscsa.edu>


Please remove my name from the pombe mailing list.  I am retiring.

Sincerely,
Larry D. Barnes

Larry D. Barnes
Associate Dean
Office of the Graduate Dean
   and the Department of Biochemistry
University of Texas Health Science Center
San Antonio, TX  78229-3900
P:  (210) 567-3711
F:  (210) 567-3719
barnesl at uthscsa.edu<mailto:barnesl at uthscsa.edu>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120703/02d42de5/attachment.htm>

From mah79 at cam.ac.uk  Tue Jul  3 16:09:35 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Tue, 3 Jul 2012 16:09:35 +0100 (BST)
Subject: [Pombelist] PomBase - flanking region sequence
In-Reply-To: <Pine.LNX.4.64.1207031118480.2194@hermes-2.csi.cam.ac.uk>
References: <Pine.LNX.4.64.1207031118480.2194@hermes-2.csi.cam.ac.uk>
Message-ID: <Pine.LNX.4.64.1207031608400.2194@hermes-2.csi.cam.ac.uk>

Dear pombe researchers,

The sequence download problem has now been corrected. Please let us know 
if you encounter any further troubles, new or old, with the PomBase site.

Midori

On Tue, 3 Jul 2012, Dr Midori A. Harris wrote:

> Dear pombe researchers,
>
> We regret to inform you that, although one bug in the Sequence Download has 
> indeed been fixed, as reported yesterday, another problem has cropped up. The 
> custom download feature is not working properly, and does not retrieve 
> flanking regions.
>
> We will alert you as soon as this problem is corrected. In the meantime, we 
> appreciate your patience.
>
> Many thanks to Charlie Hoffman for noticing and reporting this problem. 
> Please keep sending your comments, positive and negative, so that we can 
> continue to improve PomBase.
>
> Thank you,
> Midori
>
> ---------------------------
> Midori A. Harris, PhD
> Database Curator, PomBase
> Department of Biochemistry
> University of Cambridge
> Cambridge, UK
> mah79 at cam.ac.uk
>
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>


From mah79 at cam.ac.uk  Wed Jul  4 17:34:27 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Wed, 4 Jul 2012 17:34:27 +0100 (BST)
Subject: [Pombelist] The PomBase FAQ
Message-ID: <Pine.LNX.4.64.1207041732330.17517@hermes-2.csi.cam.ac.uk>

Dear pombe researchers,

Now that the PomBase web site (www.pombase.org) is past its preview phase, 
one of the things we are working on is improving documentation throughout 
the site. The first piece of documentation is now available: a Frequently 
Asked Questions (FAQ) page, where we offer assistance with doing searches, 
using the genome browser, genome regions and statistics, and much more.

To find the FAQ, follow this link:

   http://www.pombase.org/faq

Or, on any PomBase page, click the "Help" tab -- you'll reach the Help 
page, with a prominent link to the FAQ.

If you have a question you would like to have added to the FAQ, or if you 
can suggest any improvements to the questions and answers we have, please 
let us know. Email us at <helpdesk at pombase.org> any time.

Best wishes,
Midori (on behalf of the PomBase team)

-----------------------------
Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk



From mah79 at cam.ac.uk  Thu Jul  5 10:55:37 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Thu, 5 Jul 2012 10:55:37 +0100 (BST)
Subject: [Pombelist] Using the pombelist archives
Message-ID: <Pine.LNX.4.64.1207041754410.17517@hermes-2.csi.cam.ac.uk>

Dear pombe researchers,

This is a friendly reminder that this list has an archive of every message 
ever sent (except spam), dating back to December 2004:

   http://publists.sanger.ac.uk/pipermail/pombelist/

We have a few suggestions for making the list and its archive as useful as 
possible for the pombe community:

- If you have a general question about pombe, check the list archive to 
see if it has been addressed before.

- Use a descriptive subject line, to make it easier to find your message, 
and any responses, later.

- If you receive several replies, especially if any aren't copied to the 
list, it's very helpful to send a summary as a reply to your original 
question.

The archive is also linked to the list information page mentioned in the 
boilerplate at the bottom of each message.

If you have any questions about the list, please ask the PomBase curators 
at <helpdesk at pombase.org>.

Best regards,
Midori (on behalf of the PomBase team)

----------------------------
Midori A. Harris, PhD
Database Curator, PomBase
Department of Biochemistry
University of Cambridge
Cambridge, UK
mah79 at cam.ac.uk



From farinaz_1358 at yahoo.com  Sun Jul  8 08:32:14 2012
From: farinaz_1358 at yahoo.com (farinaz ghods)
Date: Sun, 8 Jul 2012 00:32:14 -0700 (PDT)
Subject: [Pombelist] Problem in Transformation
Message-ID: <1341732734.27116.YahooMailNeo@web122306.mail.ne1.yahoo.com>

Dear
Colleagues,
As a
part of a project, we are trying to?perform S.pombe(972h-) transformation.
S.pombetransformations were performed
by electroporation and LiAc according to their relative protocols. The plasmid
which was used is: pFA6MX4. 
But unfortunately
none of them were successful.We have also checked all the solution one by one.
- The
expiration date of Geneticine which we have used was passed;does it have any affect on
transformation efficiency?
- Would
you?please send us any information about the plasmid stability?
I would
very much appreciate if you could kindly share with us your valuable experiences
and suggestions on this matter.
Looking
forward to hear from you soon 
Thanking you in advance

Farinaz.J.Ghods
PhD student of Genetics,
Molecular Biology and Genetics Department,
Istanbul University/Turkey
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120708/f95ca94e/attachment.htm>

From haochen.yu at bc.biol.ethz.ch  Tue Jul 10 16:47:05 2012
From: haochen.yu at bc.biol.ethz.ch (Yu  Haochen)
Date: Tue, 10 Jul 2012 15:47:05 +0000
Subject: [Pombelist] DNA FISH in pombe
Message-ID: <CC221B17.2ADC%haochen.yu@bc.biol.ethz.ch>

Dear fission fanatics,

I am in need for some expert advice on Fluorescent in situ Hybridzation in Pombe.

I have been trying to do FISH using the protocol from the Nurse Lab Handbook. This protocol probably originated from JCS paper by Uzawa & Yanagida 1992. I am trying to detect a repeat sequence of 18bp (200-1000 copies per cell) using a Cy3-labeled oligo. So far I have not obtained specific signals, only diffusive fluorescence in the cells. I controlled for fixation and spheroblasting using immunofluorscence, and it seems that these procedures were fine. The thing I am concerned about is the efficiency of cell denaturation and hybridization condition, where I do not have a good control.

Any tips will be deeply appreciated!

Best regards,

Haochen

--
Dr. Haochen (Jerry) Yu
Institut of Biochemistry
ETH Zurich
HPM D14.1
Schafmattstr. 18
8093 Zurich, Switzerland
Phone: +41 44 632 3015
Email: haochen.yu at bc.biol.ethz.ch<mailto:haochen.yu at bc.biol.ethz.ch>


From jacqueline.hayles at cancer.org.uk  Mon Jul 16 11:54:13 2012
From: jacqueline.hayles at cancer.org.uk (Jacqueline Hayles)
Date: Mon, 16 Jul 2012 11:54:13 +0100
Subject: [Pombelist] Genetics Society Pombe Club
Message-ID: <CC29B165.2DC8%jacqueline.hayles@cancer.org.uk>

Dear All

Here is the programme for the next Pombe Club on September 18th 2012 and  I hope to see lots of you there.  Because of LRI's new security measures it would be helpful if you could let me know by the beginning of September if you intend to come so that I have a rough idea of the number of attendees.

All the best

Jacky

Jacqueline Hayles
Cell Cycle Laboratory
Cancer Research UK
London Research Institute
44, Lincoln's Inn Fields
London
WC2A 3LY
UK
Tel +44 207 269 3105
Email jacqueline.hayles at cancer.org.uk

[cid:2723F8F2-F9ED-43E8-9FA0-C7BC672657D7]

NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered in England and Wales
Company Registered Number: 4325234.
Registered Charity Number: 1089464 and Scotland SC041666
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120716/2a74dec8/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 429B74C6-7439-48E3-8D51-CCABA709AB49.png
Type: image/png
Size: 56718 bytes
Desc: 429B74C6-7439-48E3-8D51-CCABA709AB49.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120716/2a74dec8/attachment-0001.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Pombe Club Notice 16.07.12 .doc
Type: application/x-msword
Size: 25088 bytes
Desc: Pombe Club Notice 16.07.12 .doc
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120716/2a74dec8/attachment-0001.bin>

From hli41 at crimson.ua.edu  Mon Jul 16 22:33:29 2012
From: hli41 at crimson.ua.edu (Huan Li)
Date: Mon, 16 Jul 2012 16:33:29 -0500
Subject: [Pombelist] problem about pON177 plasmid
Message-ID: <CANQ9GJhPQeePxOV6LHRNWCaA-27b_qQ6metf9FPjKi50OMvrFQ@mail.gmail.com>

Dear Pombe Researchers,

 I am using the pON177 plasmid and heterozygous mutant strain derived
from Sp286 (from bioneer) to make a conditional mutant haploid strain.
After successfully transforming the pON177, I got high concentration
of sporulation and conducted the tetrad dissection on YES+G418 plate.
I streaked the visible colonies on new EMM plate. However, I happened
to notice the haploid h+ strain began to mate with each other. Did
anyone encounter this problem before when dealing with pON177 plasmid?
Is the mating caused by pON177 and how can I get rid of it?

Also, did anyone have the sequence map of pON177 plasmid and can
kindly provide it to me? Another problem I have is if there exists a
plasmid that has the same function as pON177 (Mat1-M gene) but has a
different auxotrophic marker.

Thank you so much and any reply will be deeply appreciated

Regards


Huan Li
Phd student of Department of Biology Sciences
University of Alabama, United States


From cherkasv at mail.nih.gov  Tue Jul 17 05:33:01 2012
From: cherkasv at mail.nih.gov (Cherkasova, Vera (NIH/NICHD) [E])
Date: Tue, 17 Jul 2012 00:33:01 -0400
Subject: [Pombelist] leu1-32
In-Reply-To: <CANQ9GJhPQeePxOV6LHRNWCaA-27b_qQ6metf9FPjKi50OMvrFQ@mail.gmail.com>
Message-ID: <CC2A633D.977B%cherkasv@mail.nih.gov>



Dear Pombe Community,

Does anybody know sequence of the leu1-32 allele. I was unable to find this information.

Thank you very much!


--
Vera Cherkasova, Ph.D.
Laboratory of Molecular
Growth Regulation
NICHD, NIH
6 Center Drive, Bldg. 6A, room 3A05
Bethesda, MD 20892

Tel: (301) 402-1560
FAX: (301) 496-7823
E-mail: cherkasv at mail.nih.gov







From bspe2c at bangor.ac.uk  Tue Jul 17 11:15:39 2012
From: bspe2c at bangor.ac.uk (Rebecca Elizabeth Williams)
Date: Tue, 17 Jul 2012 11:15:39 +0100
Subject: [Pombelist] Hi pombe group
Message-ID: <50053B4B.9030602@bangor.ac.uk>

Hi,

I'm a first year PhD student and am subscribed to pombelist. I have been 
having problems crossing a SPBC365.10 (arp5) deletion mutant. I was 
wondering if anybody else has encountered problems with this strain or 
smilar?

Forgive my lack of correct terminology! The strain has ade6, ura4.D18, 
leu1.32 markers, which I have crossed out, but am still having problems.

Thanks in advance!!!


Becky

-- 
Rhif Elusen Gofrestredig / Registered Charity No. 1141565

Gall y neges e-bost hon, ac unrhyw atodiadau a anfonwyd gyda hi,
gynnwys deunydd cyfrinachol ac wedi eu bwriadu i'w defnyddio'n unig
gan y sawl y cawsant eu cyfeirio ato (atynt). Os ydych wedi derbyn y
neges e-bost hon trwy gamgymeriad, rhowch wybod i'r anfonwr ar
unwaith a dil?wch y neges. Os na fwriadwyd anfon y neges atoch chi,
rhaid i chi beidio ? defnyddio, cadw neu ddatgelu unrhyw wybodaeth a
gynhwysir ynddi. Mae unrhyw farn neu safbwynt yn eiddo i'r sawl a'i
hanfonodd yn unig  ac nid yw o anghenraid yn cynrychioli barn
Prifysgol Bangor. Nid yw Prifysgol Bangor yn gwarantu
bod y neges e-bost hon neu unrhyw atodiadau yn rhydd rhag firysau neu
100% yn ddiogel. Oni bai fod hyn wedi ei ddatgan yn uniongyrchol yn
nhestun yr e-bost, nid bwriad y neges e-bost hon yw ffurfio contract
rhwymol - mae rhestr o lofnodwyr awdurdodedig ar gael o Swyddfa
Cyllid Prifysgol Bangor.  www.bangor.ac.uk

This email and any attachments may contain confidential material and
is solely for the use of the intended recipient(s).  If you have
received this email in error, please notify the sender immediately
and delete this email.  If you are not the intended recipient(s), you
must not use, retain or disclose any information contained in this
email.  Any views or opinions are solely those of the sender and do
not necessarily represent those of Bangor University.
Bangor University does not guarantee that this email or
any attachments are free from viruses or 100% secure.  Unless
expressly stated in the body of the text of the email, this email is
not intended to form a binding contract - a list of authorised
signatories is available from the Bangor University Finance
Office.  www.bangor.ac.uk
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120717/9ea96bb0/attachment.htm>

From Alison.Pidoux at ed.ac.uk  Tue Jul 17 11:32:03 2012
From: Alison.Pidoux at ed.ac.uk (Alison L Pidoux)
Date: Tue, 17 Jul 2012 11:32:03 +0100
Subject: [Pombelist] leu1-32
In-Reply-To: <CC2A633D.977B%cherkasv@mail.nih.gov>
References: <CC2A633D.977B%cherkasv@mail.nih.gov>
Message-ID: <20120717113203.56157uclt5vjwkkg@www.staffmail.ed.ac.uk>

Hi,

The info on leu1-32 is in:

pDUAL, a multipurpose, multicopy vector capable of chromosomal  
integration in fission yeast.
Matsuyama A, Shirai A, Yashiroda Y, Kamata A, Horinouchi S, Yoshida M.
Yeast. 2004 Nov;21(15):1289-305.

"we first cloned the leu1-
32 allele by amplifying the DNA fragment corresponding
to the leu1+ ORF from the AM2 strain
containing leu1-32, and identified the mutation.
Sequencing of the fragment revealed a single base
substitution (G137 > A) within the leu1+ ORF
(data not shown) leading to an amino acid substitution
(G46 > E), the position of which had previously
been roughly predicted (Keeney and Boeke,
1994; Kikuchi et al., 1988)."

Cheers,

Alison



Quoting "Cherkasova, Vera (NIH/NICHD) [E]" <cherkasv at mail.nih.gov> on  
Tue, 17 Jul 2012 00:33:01 -0400:

>
>
> Dear Pombe Community,
>
> Does anybody know sequence of the leu1-32 allele. I was unable to  
> find this information.
>
> Thank you very much!
>
>
> --
> Vera Cherkasova, Ph.D.
> Laboratory of Molecular
> Growth Regulation
> NICHD, NIH
> 6 Center Drive, Bldg. 6A, room 3A05
> Bethesda, MD 20892
>
> Tel: (301) 402-1560
> FAX: (301) 496-7823
> E-mail: cherkasv at mail.nih.gov
>
>
>
>
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>



-- 
Alison Pidoux Ph.D.
Allshire Lab,
Wellcome Trust Centre for Cell Biology,
Institute of Cell Biology,
6.4 Swann Building,
University of Edinburgh,
Mayfield Road,
Edinburgh, EH9 3JR


-- 
The University of Edinburgh is a charitable body, registered in
Scotland, with registration number SC005336.




From hoffmacs at bc.edu  Tue Jul 17 13:06:59 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Tue, 17 Jul 2012 08:06:59 -0400
Subject: [Pombelist] Hi pombe group
In-Reply-To: <50053B4B.9030602@bangor.ac.uk>
References: <50053B4B.9030602@bangor.ac.uk>
Message-ID: <9103CA2B-4D0C-45FB-8294-A5C221A814E2@bc.edu>

Can you tell us exactly what is the problem?  Is it that you do not get asci or that the spores are inviable or something else?  

We cross a lot of strains that are difficult to cross, mostly due to nutrient sensing problems.  While we used to use diploid selection with ade6-M210 and ade6-M216 alleles, we no longer do that.  Here is our current method that works extremely well for us.

1.  Make sure the two strains are freshly growing.
2. Mix 1-2 colonies worth of cells together in a small spot on an EMM plate that supports growth of both strains and let them grow together overnight.
3. Transfer the entire spot to an MEA plate at 30?C (first thing in the morning; our MEA contains 0.4% glucose because many of our strains lack fructose-1,6-bisphosphatase activity. I do not know if this may also help by providing energy for meiosis.).
4. Dissect on YES plates after 24 hours.

Good luck.

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"



On Jul 17, 2012, at 6:15 AM, Rebecca Elizabeth Williams wrote:

> Hi,
> 
> I'm a first year PhD student and am subscribed to pombelist. I have been having problems crossing a SPBC365.10 (arp5) deletion mutant. I was wondering if anybody else has encountered problems with this strain or smilar?
> 
> Forgive my lack of correct terminology! The strain has ade6, ura4.D18, leu1.32 markers, which I have crossed out, but am still having problems.
> 
> Thanks in advance!!!
> 
> 
> Becky 
> 
> --  
> Rhif Elusen Gofrestredig / Registered Charity No. 1141565
> Gall y neges e-bost hon, ac unrhyw atodiadau a anfonwyd gyda hi, gynnwys deunydd cyfrinachol ac wedi eu bwriadu i'w defnyddio'n unig gan y sawl y cawsant eu cyfeirio ato (atynt). Os ydych wedi derbyn y neges e-bost hon trwy gamgymeriad, rhowch wybod i'r anfonwr ar unwaith a dil?wch y neges. Os na fwriadwyd anfon y neges atoch chi, rhaid i chi beidio ? defnyddio, cadw neu ddatgelu unrhyw wybodaeth a gynhwysir ynddi. Mae unrhyw farn neu safbwynt yn eiddo i'r sawl a'i hanfonodd yn unig ac nid yw o anghenraid yn cynrychioli barn Prifysgol Bangor. Nid yw Prifysgol Bangor yn gwarantu bod y neges e-bost hon neu unrhyw atodiadau yn rhydd rhag firysau neu 100% yn ddiogel. Oni bai fod hyn wedi ei ddatgan yn uniongyrchol yn nhestun yr e-bost, nid bwriad y neges e-bost hon yw ffurfio contract rhwymol - mae rhestr o lofnodwyr awdurdodedig ar gael o Swyddfa Cyllid Prifysgol Bangor. www.bangor.ac.uk
> 
> This email and any attachments may contain confidential material and is solely for the use of the intended recipient(s). If you have received this email in error, please notify the sender immediately and delete this email. If you are not the intended recipient(s), you must not use, retain or disclose any information contained in this email. Any views or opinions are solely those of the sender and do not necessarily represent those of Bangor University. Bangor University does not guarantee that this email or any attachments are free from viruses or 100% secure. Unless expressly stated in the body of the text of the email, this email is not intended to form a binding contract - a list of authorised signatories is available from the Bangor University Finance Office. www.bangor.ac.uk 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120717/e6975999/attachment.htm>

From c.caetano at ucl.ac.uk  Tue Jul 24 12:27:03 2012
From: c.caetano at ucl.ac.uk (Catia M Pereira Fraga Caetano)
Date: Tue, 24 Jul 2012 12:27:03 +0100
Subject: [Pombelist] Nuclei Isolation
Message-ID: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>

Dear all

Does anyone have a protocol to isolate nuclei in pombe that works? I have
been looking and it seems that most protocols use Novozym 234, which is no
longer commercially available. Can I use Zymolase instead?

Thank you in advance.

Catia Caetano
PhD student
Rob de Bruin Group
MRC Laboratory for Molecular Cell Biology
University College London
Gower St
London
WC1E 6BT
UK



From Nick.Rhind at umassmed.edu  Tue Jul 24 15:14:29 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Tue, 24 Jul 2012 14:14:29 +0000
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
References: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
Message-ID: <74CD3876-6544-42E4-AFD7-290810CDE11C@umassmed.edu>

Hi Catia,

I can't help with nuclei isolation, but Novozym 234 is available from Sigma as Lysing Enzymes (L1412) <www.sigmaaldrich.com/catalog/product/sigma/l1412>.

Nick


On 24 Jul, 12, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:

> Dear all
> 
> Does anyone have a protocol to isolate nuclei in pombe that works? I have
> been looking and it seems that most protocols use Novozym 234, which is no
> longer commercially available. Can I use Zymolase instead?
> 
> Thank you in advance.
> 
> Catia Caetano
> PhD student
> Rob de Bruin Group
> MRC Laboratory for Molecular Cell Biology
> University College London
> Gower St
> London
> WC1E 6BT
> UK
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist



From huberman at buffalo.edu  Tue Jul 24 16:05:34 2012
From: huberman at buffalo.edu (Joel Huberman)
Date: Tue, 24 Jul 2012 11:05:34 -0400
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
References: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
Message-ID: <4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>

Dear Catia,

	Bob Givens in my lab developed a protocol that works routinely. The method involves grinding flash-frozen pombe cells under liquid nitrogen. It yields intact nuclei, suitable for many purposes. We used them both for isolation of high-molecular weight DNA containing intact replication bubbles and also for isolation of nucleosomes after MNase treatment. Our method is described in these two papers:

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen.
Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA.
BMC Res Notes. 2011 Nov 16;4(1):499.

Chromatin architectures at fission yeast transcriptional promoters and replication origins.
Givens RM, Lai WK, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J, Huberman JA, Buck MJ.
Nucleic Acids Res. 2012 May 9. [Epub ahead of print]

	If you have any questions about this procedure, please let me know.

Sincerely yours,
Joel Huberman

On Jul 24, 2012, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:

> Dear all
> 
> Does anyone have a protocol to isolate nuclei in pombe that works? I have
> been looking and it seems that most protocols use Novozym 234, which is no
> longer commercially available. Can I use Zymolase instead?
> 
> Thank you in advance.
> 
> Catia Caetano
> PhD student
> Rob de Bruin Group
> MRC Laboratory for Molecular Cell Biology
> University College London
> Gower St
> London
> WC1E 6BT
> UK
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> 

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120724/4368b5c2/attachment.htm>

From Philipp.Korber at med.uni-muenchen.de  Tue Jul 24 20:51:45 2012
From: Philipp.Korber at med.uni-muenchen.de (Philipp.Korber at med.uni-muenchen.de)
Date: Tue, 24 Jul 2012 21:51:45 +0200
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>
References: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
	<4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>
Message-ID: <5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>

Dear Catia,

Meant as an alternative to Given's and Huberman's protocol, not
competition, I just point out that we published a protocol for S. pombe
nuclei isolation in Lantermann A et al., 2009, Methods. Our protocol is
dervied from a much earlier one developed in the group of Fritz Thoma (ETH
Zurich, Switzerland) published in Bernardi et al., 1992, EMBO J.
We use zymolyase for cell lysis. In contrast to budding yeast, S. pombe
does not really show a change in OD600 upon lysis. So it is more difficult
to control for lysis efficiency. Further, one needs quite a lot more
zymolyase for lysing the same number of cells compared to budding yeast.

We have not tried Bob Given'S/Joel Huberman's protocol ourselves yet
(mainly because ours works just fine on a daily basis), but we do use cryo
grinding for preparing budding yeast extracts. Cryo grinding is for sure
cheaper than zymolyase. You should just try what works best for you.

Best wishes,

Philipp Korber

Adolf-Butenandt-Institut
University of Munich
Germany




> Dear Catia,
>
> 	Bob Givens in my lab developed a protocol that works routinely. The
> method involves grinding flash-frozen pombe cells under liquid nitrogen.
> It yields intact nuclei, suitable for many purposes. We used them both
> for isolation of high-molecular weight DNA containing intact replication
> bubbles and also for isolation of nucleosomes after MNase treatment. Our
> method is described in these two papers:
>
> Integrity of chromatin and replicating DNA in nuclei released from fission
> yeast by semi-automated grinding in liquid nitrogen.
> Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA.
> BMC Res Notes. 2011 Nov 16;4(1):499.
>
> Chromatin architectures at fission yeast transcriptional promoters and
> replication origins.
> Givens RM, Lai WK, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J,
> Huberman JA, Buck MJ.
> Nucleic Acids Res. 2012 May 9. [Epub ahead of print]
>
> 	If you have any questions about this procedure, please let me know.
>
> Sincerely yours,
> Joel Huberman
>
> On Jul 24, 2012, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:
>
>> Dear all
>>
>> Does anyone have a protocol to isolate nuclei in pombe that works? I
>> have
>> been looking and it seems that most protocols use Novozym 234, which is
>> no
>> longer commercially available. Can I use Zymolase instead?
>>
>> Thank you in advance.
>>
>> Catia Caetano
>> PhD student
>> Rob de Bruin Group
>> MRC Laboratory for Molecular Cell Biology
>> University College London
>> Gower St
>> London
>> WC1E 6BT
>> UK
>>
>>
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>>
>>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist




From S.M.Sweet at sussex.ac.uk  Wed Jul 25 10:45:42 2012
From: S.M.Sweet at sussex.ac.uk (Steve Sweet)
Date: Wed, 25 Jul 2012 09:45:42 +0000
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
References: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
	<4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>
	<5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
Message-ID: <587579DA41648440A59C1991B366CD8B0C90ABBF@EX-SHA-MBX2.ad.susx.ac.uk>

Dear Philipp,

Thanks for sending the reference; it looks like a very useful protocol for mononucleosomal DNA preparation.
I'm probably just missing something here, but does this protocol isolate nuclei?

I see that it generates spheroplasts, which are then permeabilized using detergent. Maybe I'm looking at the wrong part of the paper.

All the best,

Steve Sweet

---
Dr Steve Sweet, 
Senior Research Fellow,
Genome Damage and Stability Centre,
University of Sussex.

Tel: 01273 877 414

http://www.sussex.ac.uk/lifesci/sweetlab/



-----Original Message-----
From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Philipp.Korber at med.uni-muenchen.de
Sent: 24 July 2012 20:52
To: Joel Huberman
Cc: Bob Givens; pombelist at sanger.ac.uk; Catia M Pereira Fraga Caetano
Subject: Re: [Pombelist] Nuclei Isolation

Dear Catia,

Meant as an alternative to Given's and Huberman's protocol, not competition, I just point out that we published a protocol for S. pombe nuclei isolation in Lantermann A et al., 2009, Methods. Our protocol is dervied from a much earlier one developed in the group of Fritz Thoma (ETH Zurich, Switzerland) published in Bernardi et al., 1992, EMBO J.
We use zymolyase for cell lysis. In contrast to budding yeast, S. pombe does not really show a change in OD600 upon lysis. So it is more difficult to control for lysis efficiency. Further, one needs quite a lot more zymolyase for lysing the same number of cells compared to budding yeast.

We have not tried Bob Given'S/Joel Huberman's protocol ourselves yet (mainly because ours works just fine on a daily basis), but we do use cryo grinding for preparing budding yeast extracts. Cryo grinding is for sure cheaper than zymolyase. You should just try what works best for you.

Best wishes,

Philipp Korber

Adolf-Butenandt-Institut
University of Munich
Germany




> Dear Catia,
>
> 	Bob Givens in my lab developed a protocol that works routinely. The 
> method involves grinding flash-frozen pombe cells under liquid nitrogen.
> It yields intact nuclei, suitable for many purposes. We used them both 
> for isolation of high-molecular weight DNA containing intact 
> replication bubbles and also for isolation of nucleosomes after MNase 
> treatment. Our method is described in these two papers:
>
> Integrity of chromatin and replicating DNA in nuclei released from 
> fission yeast by semi-automated grinding in liquid nitrogen.
> Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA.
> BMC Res Notes. 2011 Nov 16;4(1):499.
>
> Chromatin architectures at fission yeast transcriptional promoters and 
> replication origins.
> Givens RM, Lai WK, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J, 
> Huberman JA, Buck MJ.
> Nucleic Acids Res. 2012 May 9. [Epub ahead of print]
>
> 	If you have any questions about this procedure, please let me know.
>
> Sincerely yours,
> Joel Huberman
>
> On Jul 24, 2012, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:
>
>> Dear all
>>
>> Does anyone have a protocol to isolate nuclei in pombe that works? I 
>> have been looking and it seems that most protocols use Novozym 234, 
>> which is no longer commercially available. Can I use Zymolase 
>> instead?
>>
>> Thank you in advance.
>>
>> Catia Caetano
>> PhD student
>> Rob de Bruin Group
>> MRC Laboratory for Molecular Cell Biology University College London 
>> Gower St London WC1E 6BT UK
>>
>>
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>>
>>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist



_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://publists.sanger.ac.uk/mailman/listinfo/pombelist


From pkorber at med.uni-muenchen.de  Wed Jul 25 11:06:26 2012
From: pkorber at med.uni-muenchen.de (Philipp Korber)
Date: Wed, 25 Jul 2012 12:06:26 +0200
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <587579DA41648440A59C1991B366CD8B0C90ABBF@EX-SHA-MBX2.ad.su
	sx.ac.uk>
References: <5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
	<bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
	<4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>
	<5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
Message-ID: <20120725100626.376921900C1@wifomail.med.uni-muenchen.de>

Dear Steve,

Thanks for your reply. Yes, you are right, our protocol is useful for
looking at chromatin, but we do not isolate nuclei as such. I am sorry for
the confusion.

The reason for this confusion on my part is actually an interesting
technical detail. We do a lot of nuclei isolation for chromatin analysis in
S. cerevisiae (classical spheroplasting with zymolyase and then osmotic
shock and centrifugation through Ficoll). At first we tried this for S.
pombe, too. However, this way we observed a lot of DNA cleavage by
endogenous nucleases. So we changed the protocol to
spheroplasting/detergent, but in analogy to our S. cerevisiae work we keep
saying in lab jargon that we do "nuclei preparation". Sorry about that.

The spheroplasting/detergent protocol that we published gives much less
endogenous cleavage. We are not sure why but guess that this protocol is
less disruptive to some other compartment (lysosome, vacuole?) which may
contain this endogenous nuclease. Any more specific answers are welcome.

Best wishes,

Philipp


At 09:45 25.07.2012 +0000, Steve Sweet wrote:
>Dear Philipp,
>
>Thanks for sending the reference; it looks like a very useful protocol for
mononucleosomal DNA preparation.
>I'm probably just missing something here, but does this protocol isolate
nuclei?
>
>I see that it generates spheroplasts, which are then permeabilized using
detergent. Maybe I'm looking at the wrong part of the paper.
>
>All the best,
>
>Steve Sweet
>
>---
>Dr Steve Sweet, 
>Senior Research Fellow,
>Genome Damage and Stability Centre,
>University of Sussex.

>
>Tel: 01273 877 414
>
>http://www.sussex.ac.uk/lifesci/sweetlab/
>
>
>
>-----Original Message-----
>From: pombelist-bounces at sanger.ac.uk
[mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of
Philipp.Korber at med.uni-muenchen.de
>Sent: 24 July 2012 20:52
>To: Joel Huberman
>Cc: Bob Givens; pombelist at sanger.ac.uk; Catia M Pereira Fraga Caetano
>Subject: Re: [Pombelist] Nuclei Isolation
>
>Dear Catia,
>
>Meant as an alternative to Given's and Huberman's protocol, not
competition, I just point out that we published a protocol for S. pombe
nuclei isolation in Lantermann A et al., 2009, Methods. Our protocol is
dervied from a much earlier one developed in the group of Fritz Thoma (ETH
Zurich, Switzerland) published in Bernardi et al., 1992, EMBO J.
>We use zymolyase for cell lysis. In contrast to budding yeast, S. pombe
does not really show a change in OD600 upon lysis. So it is more difficult
to control for lysis efficiency. Further, one needs quite a lot more
zymolyase for lysing the same number of cells compared to budding yeast.
>
>We have not tried Bob Given'S/Joel Huberman's protocol ourselves yet
(mainly because ours works just fine on a daily basis), but we do use cryo
grinding for preparing budding yeast extracts. Cryo grinding is for sure
cheaper than zymolyase. You should just try what works best for you.
>
>Best wishes,
>
>Philipp Korber
>
>Adolf-Butenandt-Institut
>University of Munich
>Germany
>
>
>
>
>> Dear Catia,
>>
>> 	Bob Givens in my lab developed a protocol that works routinely. The 
>> method involves grinding flash-frozen pombe cells under liquid nitrogen.
>> It yields intact nuclei, suitable for many purposes. We used them both 
>> for isolation of high-molecular weight DNA containing intact 
>> replication bubbles and also for isolation of nucleosomes after MNase 
>> treatment. Our method is described in these two papers:
>>
>> Integrity of chromatin and replicating DNA in nuclei released from 
>> fission yeast by semi-automated grinding in liquid nitrogen.
>> Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA.
>> BMC Res Notes. 2011 Nov 16;4(1):499.
>>
>> Chromatin architectures at fission yeast transcriptional promoters and 
>> replication origins.
>> Givens RM, Lai WK, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J, 
>> Huberman JA, Buck MJ.
>> Nucleic Acids Res. 2012 May 9. [Epub ahead of print]
>>
>> 	If you have any questions about this procedure, please let me know.
>>
>> Sincerely yours,
>> Joel Huberman
>>
>> On Jul 24, 2012, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:
>>

>>> Dear all
>>>
>>> Does anyone have a protocol to isolate nuclei in pombe that works? I 
>>> have been looking and it seems that most protocols use Novozym 234, 
>>> which is no longer commercially available. Can I use Zymolase 
>>> instead?
>>>
>>> Thank you in advance.
>>>
>>> Catia Caetano
>>> PhD student
>>> Rob de Bruin Group
>>> MRC Laboratory for Molecular Cell Biology University College London 
>>> Gower St London WC1E 6BT UK
>>>
>>>
>>> _______________________________________________
>>> Pombelist mailing list
>>> Pombelist at sanger.ac.uk
>>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>>>
>>>
>>
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>
>
>_______________________________________________
>Pombelist mailing list
>Pombelist at sanger.ac.uk
>http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 


From huberman at buffalo.edu  Wed Jul 25 14:22:47 2012
From: huberman at buffalo.edu (Joel Huberman)
Date: Wed, 25 Jul 2012 09:22:47 -0400
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <20120725100626.376921900C1@wifomail.med.uni-muenchen.de>
References: <5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
	<bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
	<4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>
	<5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
	<20120725100626.376921900C1@wifomail.med.uni-muenchen.de>
Message-ID: <B82682C5-7ADF-403F-95AD-6816F3AB63C5@buffalo.edu>

Dear Philipp and Steve,

	Let me take this opportunity to point out that our frozen-cell-grinding protocol (see my recent post to PombeList) does permit isolation of morphologically (by light microscopy) intact nuclei, free of detectable endogenous nuclease. And our protocol does not require the use of Zymolyase or other lytic enzyme. However, because our protocol employs a rather large commercial mortar grinder (Retsch RM100 or RM200), it is not suitable for nuclear isolation from small numbers of cells. In that case, if chromatin (rather than nuclei) will be sufficient, then the protocol published by Philipp would be best.

	I am currently working on editing instructional movies prepared by Bob Givens (who developed the nuclear isolation technique in my lab). These movies will show in detail all steps of our nuclear isolation procedure (and also of our high-molecular-weight DNA isolation procedure). As soon as my editing is complete and I have posted the movies to the Internet, I'll send another message to PombeList announcing that fact.

With best wishes,
Joel Huberman

On Jul 25, 2012, at 6:06 AM, Philipp Korber wrote:

> Dear Steve,
> 
> Thanks for your reply. Yes, you are right, our protocol is useful for
> looking at chromatin, but we do not isolate nuclei as such. I am sorry for
> the confusion.
> 
> The reason for this confusion on my part is actually an interesting
> technical detail. We do a lot of nuclei isolation for chromatin analysis in
> S. cerevisiae (classical spheroplasting with zymolyase and then osmotic
> shock and centrifugation through Ficoll). At first we tried this for S.
> pombe, too. However, this way we observed a lot of DNA cleavage by
> endogenous nucleases. So we changed the protocol to
> spheroplasting/detergent, but in analogy to our S. cerevisiae work we keep
> saying in lab jargon that we do "nuclei preparation". Sorry about that.
> 
> The spheroplasting/detergent protocol that we published gives much less
> endogenous cleavage. We are not sure why but guess that this protocol is
> less disruptive to some other compartment (lysosome, vacuole?) which may
> contain this endogenous nuclease. Any more specific answers are welcome.
> 
> Best wishes,
> 
> Philipp
> 
> 
> At 09:45 25.07.2012 +0000, Steve Sweet wrote:
>> Dear Philipp,
>> 
>> Thanks for sending the reference; it looks like a very useful protocol for
> mononucleosomal DNA preparation.
>> I'm probably just missing something here, but does this protocol isolate
> nuclei?
>> 
>> I see that it generates spheroplasts, which are then permeabilized using
> detergent. Maybe I'm looking at the wrong part of the paper.
>> 
>> All the best,
>> 
>> Steve Sweet
>> 
>> ---
>> Dr Steve Sweet, 
>> Senior Research Fellow,
>> Genome Damage and Stability Centre,
>> University of Sussex.
> 
>> 
>> Tel: 01273 877 414
>> 
>> http://www.sussex.ac.uk/lifesci/sweetlab/
>> 
>> 
>> 
>> -----Original Message-----
>> From: pombelist-bounces at sanger.ac.uk
> [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of
> Philipp.Korber at med.uni-muenchen.de
>> Sent: 24 July 2012 20:52
>> To: Joel Huberman
>> Cc: Bob Givens; pombelist at sanger.ac.uk; Catia M Pereira Fraga Caetano
>> Subject: Re: [Pombelist] Nuclei Isolation
>> 
>> Dear Catia,
>> 
>> Meant as an alternative to Given's and Huberman's protocol, not
> competition, I just point out that we published a protocol for S. pombe
> nuclei isolation in Lantermann A et al., 2009, Methods. Our protocol is
> dervied from a much earlier one developed in the group of Fritz Thoma (ETH
> Zurich, Switzerland) published in Bernardi et al., 1992, EMBO J.
>> We use zymolyase for cell lysis. In contrast to budding yeast, S. pombe
> does not really show a change in OD600 upon lysis. So it is more difficult
> to control for lysis efficiency. Further, one needs quite a lot more
> zymolyase for lysing the same number of cells compared to budding yeast.
>> 
>> We have not tried Bob Given'S/Joel Huberman's protocol ourselves yet
> (mainly because ours works just fine on a daily basis), but we do use cryo
> grinding for preparing budding yeast extracts. Cryo grinding is for sure
> cheaper than zymolyase. You should just try what works best for you.
>> 
>> Best wishes,
>> 
>> Philipp Korber
>> 
>> Adolf-Butenandt-Institut
>> University of Munich
>> Germany
>> 
>> 
>> 
>> 
>>> Dear Catia,
>>> 
>>> 	Bob Givens in my lab developed a protocol that works routinely. The 
>>> method involves grinding flash-frozen pombe cells under liquid nitrogen.
>>> It yields intact nuclei, suitable for many purposes. We used them both 
>>> for isolation of high-molecular weight DNA containing intact 
>>> replication bubbles and also for isolation of nucleosomes after MNase 
>>> treatment. Our method is described in these two papers:
>>> 
>>> Integrity of chromatin and replicating DNA in nuclei released from 
>>> fission yeast by semi-automated grinding in liquid nitrogen.
>>> Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA.
>>> BMC Res Notes. 2011 Nov 16;4(1):499.
>>> 
>>> Chromatin architectures at fission yeast transcriptional promoters and 
>>> replication origins.
>>> Givens RM, Lai WK, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J, 
>>> Huberman JA, Buck MJ.
>>> Nucleic Acids Res. 2012 May 9. [Epub ahead of print]
>>> 
>>> 	If you have any questions about this procedure, please let me know.
>>> 
>>> Sincerely yours,
>>> Joel Huberman
>>> 
>>> On Jul 24, 2012, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:
>>> 
> 
>>>> Dear all
>>>> 
>>>> Does anyone have a protocol to isolate nuclei in pombe that works? I 
>>>> have been looking and it seems that most protocols use Novozym 234, 
>>>> which is no longer commercially available. Can I use Zymolase 
>>>> instead?
>>>> 
>>>> Thank you in advance.
>>>> 
>>>> Catia Caetano
>>>> PhD student
>>>> Rob de Bruin Group
>>>> MRC Laboratory for Molecular Cell Biology University College London 
>>>> Gower St London WC1E 6BT UK
>>>> 
>>>> 
>>>> _______________________________________________
>>>> Pombelist mailing list
>>>> Pombelist at sanger.ac.uk
>>>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>>>> 
>>>> 
>>> 
>>> _______________________________________________
>>> Pombelist mailing list
>>> Pombelist at sanger.ac.uk
>>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>> 
>> 
>> 
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> 



From punitprasad at gmail.com  Wed Jul 25 15:10:22 2012
From: punitprasad at gmail.com (Punit Prasad)
Date: Wed, 25 Jul 2012 16:10:22 +0200
Subject: [Pombelist] Nuclei Isolation
In-Reply-To: <B82682C5-7ADF-403F-95AD-6816F3AB63C5@buffalo.edu>
References: <bdc1a1c863f9a2d1c03c7c3d45c0b1a1.squirrel@www.squirrelmail.ucl.ac.uk>
	<4566E48C-DA87-43C7-A2AD-2F50368FBDD7@buffalo.edu>
	<5f6d5f2180e2461a06dafea2ace93797.squirrel@wifomail.med.uni-muenchen.de>
	<20120725100626.376921900C1@wifomail.med.uni-muenchen.de>
	<B82682C5-7ADF-403F-95AD-6816F3AB63C5@buffalo.edu>
Message-ID: <CAOm0vziN+i6eeNE3hr=naW9LS46uLzP_acdgQWOTqkC=tV_qcA@mail.gmail.com>

Hello,
We have been trying to isolate nuclei and have used recently Freezer Mill
6850. In two cycles of 2min each with 2min pause and we found quite
efficient nuclei (85%) in solution as seen from DAPI staining. Since we are
planning to isolate nuclei in large scale this method we have found far
more efficient than Zymolase treatment. We have tried with 5g of frozen but
this machine can be use to grind from 100mg to 40gm of cells depending on
the size vials.
Bests
Punit

On Wed, Jul 25, 2012 at 3:22 PM, Joel Huberman <huberman at buffalo.edu> wrote:

> Dear Philipp and Steve,
>
>         Let me take this opportunity to point out that our
> frozen-cell-grinding protocol (see my recent post to PombeList) does permit
> isolation of morphologically (by light microscopy) intact nuclei, free of
> detectable endogenous nuclease. And our protocol does not require the use
> of Zymolyase or other lytic enzyme. However, because our protocol employs a
> rather large commercial mortar grinder (Retsch RM100 or RM200), it is not
> suitable for nuclear isolation from small numbers of cells. In that case,
> if chromatin (rather than nuclei) will be sufficient, then the protocol
> published by Philipp would be best.
>
>         I am currently working on editing instructional movies prepared by
> Bob Givens (who developed the nuclear isolation technique in my lab). These
> movies will show in detail all steps of our nuclear isolation procedure
> (and also of our high-molecular-weight DNA isolation procedure). As soon as
> my editing is complete and I have posted the movies to the Internet, I'll
> send another message to PombeList announcing that fact.
>
> With best wishes,
> Joel Huberman
>
> On Jul 25, 2012, at 6:06 AM, Philipp Korber wrote:
>
> > Dear Steve,
> >
> > Thanks for your reply. Yes, you are right, our protocol is useful for
> > looking at chromatin, but we do not isolate nuclei as such. I am sorry
> for
> > the confusion.
> >
> > The reason for this confusion on my part is actually an interesting
> > technical detail. We do a lot of nuclei isolation for chromatin analysis
> in
> > S. cerevisiae (classical spheroplasting with zymolyase and then osmotic
> > shock and centrifugation through Ficoll). At first we tried this for S.
> > pombe, too. However, this way we observed a lot of DNA cleavage by
> > endogenous nucleases. So we changed the protocol to
> > spheroplasting/detergent, but in analogy to our S. cerevisiae work we
> keep
> > saying in lab jargon that we do "nuclei preparation". Sorry about that.
> >
> > The spheroplasting/detergent protocol that we published gives much less
> > endogenous cleavage. We are not sure why but guess that this protocol is
> > less disruptive to some other compartment (lysosome, vacuole?) which may
> > contain this endogenous nuclease. Any more specific answers are welcome.
> >
> > Best wishes,
> >
> > Philipp
> >
> >
> > At 09:45 25.07.2012 +0000, Steve Sweet wrote:
> >> Dear Philipp,
> >>
> >> Thanks for sending the reference; it looks like a very useful protocol
> for
> > mononucleosomal DNA preparation.
> >> I'm probably just missing something here, but does this protocol isolate
> > nuclei?
> >>
> >> I see that it generates spheroplasts, which are then permeabilized using
> > detergent. Maybe I'm looking at the wrong part of the paper.
> >>
> >> All the best,
> >>
> >> Steve Sweet
> >>
> >> ---
> >> Dr Steve Sweet,
> >> Senior Research Fellow,
> >> Genome Damage and Stability Centre,
> >> University of Sussex.
> >
> >>
> >> Tel: 01273 877 414
> >>
> >> http://www.sussex.ac.uk/lifesci/sweetlab/
> >>
> >>
> >>
> >> -----Original Message-----
> >> From: pombelist-bounces at sanger.ac.uk
> > [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of
> > Philipp.Korber at med.uni-muenchen.de
> >> Sent: 24 July 2012 20:52
> >> To: Joel Huberman
> >> Cc: Bob Givens; pombelist at sanger.ac.uk; Catia M Pereira Fraga Caetano
> >> Subject: Re: [Pombelist] Nuclei Isolation
> >>
> >> Dear Catia,
> >>
> >> Meant as an alternative to Given's and Huberman's protocol, not
> > competition, I just point out that we published a protocol for S. pombe
> > nuclei isolation in Lantermann A et al., 2009, Methods. Our protocol is
> > dervied from a much earlier one developed in the group of Fritz Thoma
> (ETH
> > Zurich, Switzerland) published in Bernardi et al., 1992, EMBO J.
> >> We use zymolyase for cell lysis. In contrast to budding yeast, S. pombe
> > does not really show a change in OD600 upon lysis. So it is more
> difficult
> > to control for lysis efficiency. Further, one needs quite a lot more
> > zymolyase for lysing the same number of cells compared to budding yeast.
> >>
> >> We have not tried Bob Given'S/Joel Huberman's protocol ourselves yet
> > (mainly because ours works just fine on a daily basis), but we do use
> cryo
> > grinding for preparing budding yeast extracts. Cryo grinding is for sure
> > cheaper than zymolyase. You should just try what works best for you.
> >>
> >> Best wishes,
> >>
> >> Philipp Korber
> >>
> >> Adolf-Butenandt-Institut
> >> University of Munich
> >> Germany
> >>
> >>
> >>
> >>
> >>> Dear Catia,
> >>>
> >>>     Bob Givens in my lab developed a protocol that works routinely. The
> >>> method involves grinding flash-frozen pombe cells under liquid
> nitrogen.
> >>> It yields intact nuclei, suitable for many purposes. We used them both
> >>> for isolation of high-molecular weight DNA containing intact
> >>> replication bubbles and also for isolation of nucleosomes after MNase
> >>> treatment. Our method is described in these two papers:
> >>>
> >>> Integrity of chromatin and replicating DNA in nuclei released from
> >>> fission yeast by semi-automated grinding in liquid nitrogen.
> >>> Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA.
> >>> BMC Res Notes. 2011 Nov 16;4(1):499.
> >>>
> >>> Chromatin architectures at fission yeast transcriptional promoters and
> >>> replication origins.
> >>> Givens RM, Lai WK, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J,
> >>> Huberman JA, Buck MJ.
> >>> Nucleic Acids Res. 2012 May 9. [Epub ahead of print]
> >>>
> >>>     If you have any questions about this procedure, please let me know.
> >>>
> >>> Sincerely yours,
> >>> Joel Huberman
> >>>
> >>> On Jul 24, 2012, at 7:27 AM, Catia M Pereira Fraga Caetano wrote:
> >>>
> >
> >>>> Dear all
> >>>>
> >>>> Does anyone have a protocol to isolate nuclei in pombe that works? I
> >>>> have been looking and it seems that most protocols use Novozym 234,
> >>>> which is no longer commercially available. Can I use Zymolase
> >>>> instead?
> >>>>
> >>>> Thank you in advance.
> >>>>
> >>>> Catia Caetano
> >>>> PhD student
> >>>> Rob de Bruin Group
> >>>> MRC Laboratory for Molecular Cell Biology University College London
> >>>> Gower St London WC1E 6BT UK
> >>>>
> >>>>
> >>>> _______________________________________________
> >>>> Pombelist mailing list
> >>>> Pombelist at sanger.ac.uk
> >>>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> >>>>
> >>>>
> >>>
> >>> _______________________________________________
> >>> Pombelist mailing list
> >>> Pombelist at sanger.ac.uk
> >>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> >>
> >>
> >>
> >> _______________________________________________
> >> Pombelist mailing list
> >> Pombelist at sanger.ac.uk
> >> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> >>
> >
> > _______________________________________________
> > Pombelist mailing list
> > Pombelist at sanger.ac.uk
> > http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> >
> >
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>



-- 
*Punit Prasad*
Post Doctoral Fellow
Dept of Bioscience and Nutrition
Karolinska Institute
Stockholm
Sweden
http://www.bionut.ki.se/groups/kek/
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120725/71dbc3b5/attachment-0001.htm>

From byg2013 at nottingham.ac.uk  Wed Jul 25 16:14:57 2012
From: byg2013 at nottingham.ac.uk (Pl-Byg2013)
Date: Wed, 25 Jul 2012 16:14:57 +0100
Subject: [Pombelist] Bristish Yeast Group 2013
In-Reply-To: <069F34ED-90B1-4C61-972B-915F3A8ED7AC@exmail.nottingham.ac.uk>
References: <069F34ED-90B1-4C61-972B-915F3A8ED7AC@exmail.nottingham.ac.uk>
Message-ID: <4C2A36175EB9494E9E2CE7A3F43E156420B96D1994@EXCHANGE1.ad.nottingham.ac.uk>

Dear yeast researcher,

The 2013 British Yeast Group meeting (BYG2013) will be held in Nottingham from Wednesday 20th through to Friday 22nd March 2013. The keynote lecture will be delivered by Prof. Charlie Boone. Further details will be available in September.

Please note this date in your diary. We look forward to welcoming you to Nottingham.

Best wishes,

Conrad Nieduszynski & Carolin M?ller

Centre for Genetics and Genomics
University of Nottingham

PS - To remove yourself from this mailing list please respond to this e-mail with the word 'remove' in the subject line.
This message and any attachment are intended solely for the addressee and may contain confidential information. If you have received this message in error, please send it back to me, and immediately delete it.   Please do not use, copy or disclose the information contained in this message or in any attachment.  Any views or opinions expressed by the author of this email do not necessarily reflect the views of the University of Nottingham.

This message has been checked for viruses but the contents of an attachment
may still contain software viruses which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120725/a9f26ea7/attachment.htm>

From liyanjian at gmail.com  Thu Aug  2 14:17:07 2012
From: liyanjian at gmail.com (=?GB2312?B?wO7R172j?=)
Date: Thu, 2 Aug 2012 21:17:07 +0800
Subject: [Pombelist] Ask for elutriation protocal
Message-ID: <CAKfkQLTdBDGF3eLUfJewhO05EDxsQnksm5Ty=4QmnJ2qhCDQaw@mail.gmail.com>

Hi all,
These days our lab was trying to use elutriation to synchronize pombe, but
we have never done this experiment. Does anyone can offer us an elutriation
protocal for pombe?
Thank you very much.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120802/4193bebe/attachment.htm>

From a.m.carr at sussex.ac.uk  Thu Aug  2 14:29:14 2012
From: a.m.carr at sussex.ac.uk (Antony Carr)
Date: Thu, 2 Aug 2012 13:29:14 +0000
Subject: [Pombelist] Ask for elutriation protocal
In-Reply-To: <CAKfkQLTdBDGF3eLUfJewhO05EDxsQnksm5Ty=4QmnJ2qhCDQaw@mail.gmail.com>
References: <CAKfkQLTdBDGF3eLUfJewhO05EDxsQnksm5Ty=4QmnJ2qhCDQaw@mail.gmail.com>
Message-ID: <C5AD4BE27ED4F042BF57EA254C30BC223421A170@EX-SHA-MBX1.ad.susx.ac.uk>

Here is a chapter we wrote a long time ago for methods enzymology.


Tony Carr
Director, GDSC
University of Sussex
01273 678122

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of ???
Sent: 02 August 2012 14:17
To: pombelist at sanger.ac.uk
Subject: [Pombelist] Ask for elutriation protocal

Hi all,
These days our lab was trying to use elutriation to synchronize pombe, but we have never done this experiment. Does anyone can offer us an elutriation protocal for pombe?
Thank you very much.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120802/104d6fab/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: REVIEW3.DOC
Type: application/msword
Size: 75776 bytes
Desc: REVIEW3.DOC
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120802/104d6fab/attachment-0001.wiz>

From Nick.Rhind at umassmed.edu  Thu Aug  2 15:03:49 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Thu, 2 Aug 2012 14:03:49 +0000
Subject: [Pombelist] Ask for elutriation protocal
In-Reply-To: <C5AD4BE27ED4F042BF57EA254C30BC223421A170@EX-SHA-MBX1.ad.susx.ac.uk>
References: <CAKfkQLTdBDGF3eLUfJewhO05EDxsQnksm5Ty=4QmnJ2qhCDQaw@mail.gmail.com>
	<C5AD4BE27ED4F042BF57EA254C30BC223421A170@EX-SHA-MBX1.ad.susx.ac.uk>
Message-ID: <C9175DD8-448A-446B-9113-A63AC44E1CDE@umassmed.edu>

Here is a similar chapter from a recent issues of Methods in Molecular Biology.

Nick


On 2 Aug, 12, at 9:29 AM, Antony Carr wrote:

> Here is a chapter we wrote a long time ago for methods enzymology.
>  
>  
> Tony Carr
> Director, GDSC
> University of Sussex
> 01273 678122
>  
> From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of ???
> Sent: 02 August 2012 14:17
> To: pombelist at sanger.ac.uk
> Subject: [Pombelist] Ask for elutriation protocal
>  
> Hi all,
> These days our lab was trying to use elutriation to synchronize pombe, but we have never done this experiment. Does anyone can offer us an elutriation protocal for pombe?
> Thank you very much.
> <REVIEW3.DOC>_______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Willis & Rhind (2011) MiMB Chpt 1.pdf
Type: application/pdf
Size: 379401 bytes
Desc: Willis & Rhind (2011) MiMB Chpt 1.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120802/f8acda40/attachment-0001.pdf>

From s.codlin at ucl.ac.uk  Thu Aug  2 15:49:42 2012
From: s.codlin at ucl.ac.uk (Codlin, Sandra)
Date: Thu, 2 Aug 2012 14:49:42 +0000
Subject: [Pombelist] plate pourers
Message-ID: <04573AD8-D4BE-43E4-BEBE-1AA8AC73B603@ucl.ac.uk>

Hi,
Would anybody have experience with plate pourers for 90mm petri plates (for sterile YES agar plates etc and maybe even for ROTOR plates) and can recommend a good company/machine.

Many thanks in advance.

Best wishes,
Sandra

Sandra Codlin, PhD
Lab Manager of J*rg Bahler's Systems Biology Group,
Department of Genetics, Evolution and Environment,
The Darwin Building,
University College London
London WC1E 6BT, UK
-------------------------------------
s.codlin at ucl.ac.uk<mailto:s.codlin at ucl.ac.uk>
Phone: +44(0)203-108-1605
Ext: 51605
Fax: +44(0)207-679-7096
www.ucl.ac.uk/bahlerlab<http://www.ucl.ac.uk/bahlerlab>





-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120802/68b65559/attachment.htm>

From Harry at singerinstruments.com  Thu Aug  2 16:16:17 2012
From: Harry at singerinstruments.com (Harry Singer)
Date: Thu, 2 Aug 2012 15:16:17 +0000
Subject: [Pombelist] plate pourers
In-Reply-To: <04573AD8-D4BE-43E4-BEBE-1AA8AC73B603@ucl.ac.uk>
References: <04573AD8-D4BE-43E4-BEBE-1AA8AC73B603@ucl.ac.uk>
Message-ID: <FF6E1FE1-FB69-44A1-93BF-6F4E6DCC7997@singerinstruments.com>

Hi Sandra,
We're in the process of developing one. Small footprint, 50 plate capacity, all format round petri (inc 90mm) and square format RoToR PlusPlates. Want to beta-test one of our second-gen prototypes?
Harry

Singer Instruments
www.singerinstruments.com<http://www.singerinstruments.com>



On 2 Aug 2012, at 10:52, "Codlin, Sandra" <s.codlin at ucl.ac.uk<mailto:s.codlin at ucl.ac.uk>> wrote:

Hi,
Would anybody have experience with plate pourers for 90mm petri plates (for sterile YES agar plates etc and maybe even for ROTOR plates) and can recommend a good company/machine.

Many thanks in advance.

Best wishes,
Sandra

Sandra Codlin, PhD
Lab Manager of J*rg Bahler's Systems Biology Group,
Department of Genetics, Evolution and Environment,
The Darwin Building,
University College London
London WC1E 6BT, UK
-------------------------------------
s.codlin at ucl.ac.uk<mailto:s.codlin at ucl.ac.uk>
Phone: +44(0)203-108-1605
Ext: 51605
Fax: +44(0)207-679-7096
www.ucl.ac.uk/bahlerlab<http://www.ucl.ac.uk/bahlerlab>





_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://publists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120802/5e9e7a08/attachment.htm>

From MEYERLF at bc.edu  Tue Aug 14 17:04:03 2012
From: MEYERLF at bc.edu (Lauren Meyer)
Date: Tue, 14 Aug 2012 12:04:03 -0400
Subject: [Pombelist] FACS with S. pombe spores
Message-ID: <web-54766526@be1.bc.edu>

Hi All,

Does anybody have a protocol that they have successfully used to measure GFP signal in S. pombe spores using FACS analysis? Any FACS protocol for spores would be helpful, even if it was not measuring for GFP specifically. 

Thank you,
Lauren Meyer

Boston College


From yjli at uchicago.edu  Wed Aug 15 16:52:58 2012
From: yjli at uchicago.edu (Yujie Li)
Date: Wed, 15 Aug 2012 10:52:58 -0500
Subject: [Pombelist] pSGP572 expression vector
Message-ID: <51DEB476-1B13-4940-80FD-DC66FE65D5CA@uchicago.edu>

Hi All,

Does anybody have a pSGP572 S. pombe expression vector, which has a  
Pnmt-41x promoter and C-terminal GFP? I have this vector with inserts,  
but it would be great to have an empty vector as a negative control  
for gene over-expression.

Thank you,
Yujie Li

University of Chicago


From marc.elgort at biochem.utah.edu  Thu Aug 30 20:18:01 2012
From: marc.elgort at biochem.utah.edu (Marc Geoffrey Elgort)
Date: Thu, 30 Aug 2012 19:18:01 +0000
Subject: [Pombelist] generating point mutants in diploid strains
Message-ID: <7646EFDD587C144D888CC29EBA94A5D1035EB748@X-MB12.xds.umail.utah.edu>

Hi all

I'm trying to generate point mutations in an essential gene, so I interrupted one copy of the gene in a diploid with URA4 selected on -ura  and used PCR to verify disruption of the native locus. This all worked fine. Next, I transformed the exon containing the mutation into these strains and selected on 5FOA and used PCR to a) verify the intact locus and b) validate that URA4 was gone. This too worked fine. My next step is where things went awry: I PCRd up a fragment containing the intron-exon junctions at either end of the exon I had replaced to verify that a) recombination didn't screw things up and b) to verify the mutations within the exon by sequencing. Everything was clean except that the gene is wild type in all cases. I sequenced 3-4 clones of each of 3 mutations and in all cases I got back wild type. Is sister chromatid repair that good or is there something I'm missing? Any assistance would be greatly appreciated.

Thanks! 

Marc


Marc Elgort, PhD
Frost Laboratory
University of Utah Department of Biochemistry
15 N Medical Dr East Rm 4100
Salt Lake City, UT 84112-5650
801.581.4832




From hoffmacs at bc.edu  Thu Aug 30 20:26:28 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 30 Aug 2012 15:26:28 -0400
Subject: [Pombelist] generating point mutants in diploid strains
In-Reply-To: <7646EFDD587C144D888CC29EBA94A5D1035EB748@X-MB12.xds.umail.utah.edu>
References: <7646EFDD587C144D888CC29EBA94A5D1035EB748@X-MB12.xds.umail.utah.edu>
Message-ID: <969E849C-4227-4B23-8CC2-DA84645D217B@bc.edu>

Did you try a mock transformation to see if your transformation actually increased the frequency of 5FOA-resistant colonies when you did the knock-in?  You may need to first make sure that the homologous integration of the mutant allele is happening at a higher frequency than gene conversion of the disrupted allele to the wild type.

How much homology did you have to either side of the mutation?  What transformation protocol did you use?

Cheers,

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"



On Aug 30, 2012, at 3:18 PM, Marc Geoffrey Elgort wrote:

> Hi all
> 
> I'm trying to generate point mutations in an essential gene, so I interrupted one copy of the gene in a diploid with URA4 selected on -ura  and used PCR to verify disruption of the native locus. This all worked fine. Next, I transformed the exon containing the mutation into these strains and selected on 5FOA and used PCR to a) verify the intact locus and b) validate that URA4 was gone. This too worked fine. My next step is where things went awry: I PCRd up a fragment containing the intron-exon junctions at either end of the exon I had replaced to verify that a) recombination didn't screw things up and b) to verify the mutations within the exon by sequencing. Everything was clean except that the gene is wild type in all cases. I sequenced 3-4 clones of each of 3 mutations and in all cases I got back wild type. Is sister chromatid repair that good or is there something I'm missing? Any assistance would be greatly appreciated.
> 
> Thanks! 
> 
> Marc
> 
> 
> Marc Elgort, PhD
> Frost Laboratory
> University of Utah Department of Biochemistry
> 15 N Medical Dr East Rm 4100
> Salt Lake City, UT 84112-5650
> 801.581.4832
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist





From winston at genetics.med.harvard.edu  Thu Aug 30 21:16:56 2012
From: winston at genetics.med.harvard.edu (Fred Winston)
Date: Thu, 30 Aug 2012 16:16:56 -0400
Subject: [Pombelist] generating point mutants in diploid strains
In-Reply-To: <7646EFDD587C144D888CC29EBA94A5D1035EB748@X-MB12.xds.umail.utah.edu>
References: <7646EFDD587C144D888CC29EBA94A5D1035EB748@X-MB12.xds.umail.utah.edu>
Message-ID: <8D0B6367-8358-438B-9E90-A39C69C890FB@genetics.med.harvard.edu>

HI Marc,

If your gene is far from a centromere, a mitotic crossover between non-sister chromatids anywhere between the cen and your gene will generate a wild-type homozygote when crossing over occurs after replication and before segregation.  This type of event can obscure finding what you want.  One way we have gotten around this is in S. cerevisiae is to put a resistance marker, such as G418, next door to the URA3 insert.  Transformants that have integrated the construct will be 5FOA resistant and still G418 resistant, whereas the mitotic recombinants will be G418 sensitive.  

Fred Winston



On Aug 30, 2012, at 3:18 PM, Marc Geoffrey Elgort wrote:

> Hi all
> 
> I'm trying to generate point mutations in an essential gene, so I interrupted one copy of the gene in a diploid with URA4 selected on -ura  and used PCR to verify disruption of the native locus. This all worked fine. Next, I transformed the exon containing the mutation into these strains and selected on 5FOA and used PCR to a) verify the intact locus and b) validate that URA4 was gone. This too worked fine. My next step is where things went awry: I PCRd up a fragment containing the intron-exon junctions at either end of the exon I had replaced to verify that a) recombination didn't screw things up and b) to verify the mutations within the exon by sequencing. Everything was clean except that the gene is wild type in all cases. I sequenced 3-4 clones of each of 3 mutations and in all cases I got back wild type. Is sister chromatid repair that good or is there something I'm missing? Any assistance would be greatly appreciated.
> 
> Thanks! 
> 
> Marc
> 
> 
> Marc Elgort, PhD
> Frost Laboratory
> University of Utah Department of Biochemistry
> 15 N Medical Dr East Rm 4100
> Salt Lake City, UT 84112-5650
> 801.581.4832
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 



From marc.elgort at biochem.utah.edu  Thu Aug 30 21:21:41 2012
From: marc.elgort at biochem.utah.edu (Marc Geoffrey Elgort)
Date: Thu, 30 Aug 2012 20:21:41 +0000
Subject: [Pombelist] generating point mutants in diploid strains
In-Reply-To: <8D0B6367-8358-438B-9E90-A39C69C890FB@genetics.med.harvard.edu>
References: <7646EFDD587C144D888CC29EBA94A5D1035EB748@X-MB12.xds.umail.utah.edu>
	<8D0B6367-8358-438B-9E90-A39C69C890FB@genetics.med.harvard.edu>
Message-ID: <7646EFDD587C144D888CC29EBA94A5D1035EC7A1@X-MB12.xds.umail.utah.edu>

Thanks for the help! I think what I'm going to do is to build tagged version of the mutants and just knock them into the Leu1 locus after knocking out a single copy of the native locus. That seems more straightforward and then tracking the mutants later will be more straightforward.

Anyway, thanks again and thanks to the message board for existing, this has been infinitely helpful!

marc

-----Original Message-----
From: Fred Winston [mailto:winston at genetics.med.harvard.edu] 
Sent: Thursday, August 30, 2012 2:17 PM
To: Marc Geoffrey Elgort
Cc: pombelist at sanger.ac.uk
Subject: Re: [Pombelist] generating point mutants in diploid strains

HI Marc,

If your gene is far from a centromere, a mitotic crossover between non-sister chromatids anywhere between the cen and your gene will generate a wild-type homozygote when crossing over occurs after replication and before segregation.  This type of event can obscure finding what you want.  One way we have gotten around this is in S. cerevisiae is to put a resistance marker, such as G418, next door to the URA3 insert.  Transformants that have integrated the construct will be 5FOA resistant and still G418 resistant, whereas the mitotic recombinants will be G418 sensitive.  

Fred Winston



On Aug 30, 2012, at 3:18 PM, Marc Geoffrey Elgort wrote:

> Hi all
> 
> I'm trying to generate point mutations in an essential gene, so I interrupted one copy of the gene in a diploid with URA4 selected on -ura  and used PCR to verify disruption of the native locus. This all worked fine. Next, I transformed the exon containing the mutation into these strains and selected on 5FOA and used PCR to a) verify the intact locus and b) validate that URA4 was gone. This too worked fine. My next step is where things went awry: I PCRd up a fragment containing the intron-exon junctions at either end of the exon I had replaced to verify that a) recombination didn't screw things up and b) to verify the mutations within the exon by sequencing. Everything was clean except that the gene is wild type in all cases. I sequenced 3-4 clones of each of 3 mutations and in all cases I got back wild type. Is sister chromatid repair that good or is there something I'm missing? Any assistance would be greatly appreciated.
> 
> Thanks! 
> 
> Marc
> 
> 
> Marc Elgort, PhD
> Frost Laboratory
> University of Utah Department of Biochemistry
> 15 N Medical Dr East Rm 4100
> Salt Lake City, UT 84112-5650
> 801.581.4832
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 



From J.Millar at warwick.ac.uk  Tue Sep  4 11:27:26 2012
From: J.Millar at warwick.ac.uk (Millar, Jonathan)
Date: Tue, 4 Sep 2012 10:27:26 +0000
Subject: [Pombelist] Three post-doctoral positions: University of Warwick
Message-ID: <43D22EAB-CCFD-4F24-94FF-9398AF36CC80@live.warwick.ac.uk>

Dear All,

I would appreciate it if you could bring the attached advert for three post-doctoral positions at the University of Warwick to study "Mechanisms of spindle checkpoint silencing" in fission yeast (2 posts) and mammalian cells (1 post) to the attention of talented PhD and post-doctoral fellows. These positions are available from 1st January 2013 and are funded by the Medical Research Council (MRC) for 5 years. We are particularly interested in those individuals who have experience in either protein purification, biochemistry, mass spectrometry or quantitative live cell imaging. For more details please visit our divisional and personal websites

Division of Biomedical Cell Biology (http://www2.warwick.ac.uk/fac/med/research/biomedical/)

Professor Jonathan Millar (http://www2.warwick.ac.uk/fac/med/research/biomedical/researchareas/cellcyclecontrollab/)

Dr. Andrew McAinsh (http://mechanochemistry.org/mcainsh/)

Thank you for your time

Best wishes

Jonathan


Professor Jonathan B.A. Millar,
Division of Biomedical Cell Biology,
Warwick Medical School,
University of Warwick,
Coventry CV4 7Al, UK.
Tel: 44 247 6150 414 (office)
Tel: 44 247 6150 592 (Lab.)
Fax: 44 247 6574 637
http://www2.warwick.ac.uk/fac/med/staff/jmillar
J.Millar at warwick.ac.uk<mailto:J.Millar at warwick.ac.uk>

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120904/6b2c2773/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Post-doc. Advert.pdf
Type: application/pdf
Size: 710919 bytes
Desc: Post-doc. Advert.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120904/6b2c2773/attachment-0001.pdf>

From laharireddybiot at gmail.com  Mon Sep 10 14:08:53 2012
From: laharireddybiot at gmail.com (lahari reddy)
Date: Mon, 10 Sep 2012 18:38:53 +0530
Subject: [Pombelist] s.pombe cell cycle
Message-ID: <CANsKWwt4ORecf65OjEjyy1JOdgkszcJxUTO2H4tKYSDxAUK0+A@mail.gmail.com>

Dear all,

I have to check the cell cycle profile of a deletion mutant (say gene
related to S phase progession) that has been transformed with a suppressor
gene. The suppressor transformed strains were starved of nitrogen
(EMM-NH4Cl), but the FACS profile shows 15% of cells arrested in G1. The
plasmid  carries a uracil marker, so I thought may be the nitrogen from
uracil may hinder the arrest.

I would be pleased if you could clarify some of my doubts.

1. Is the experimental approach discussed above can be done at all, because
I didnt find evidences of articles discussing the FACS profile of a strain
that has been transformed with an ectopic plamsid in s.pombe.(pardon me if
didnt give a proper serach)

2. I thought of alternative strategy where by my deletion strain has
cdc25-22 background, but Im starting to worry whether transformation of
this temerature sensitive strain works at all with lithium acetate
protocol(growing @ 320C ) Even if plasmid can be transformed by
electroporation, Whether arrest at higher temperature and  release at lower
temperature affect the plasmid expression in  strains.



Thank you


Lahari Reddy
Graduate student
Institute of Life sciences
Hyderabad
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120910/9b4419e5/attachment.html>

From Nick.Rhind at umassmed.edu  Mon Sep 10 16:55:09 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Mon, 10 Sep 2012 15:55:09 +0000
Subject: [Pombelist] s.pombe cell cycle
In-Reply-To: <CANsKWwt4ORecf65OjEjyy1JOdgkszcJxUTO2H4tKYSDxAUK0+A@mail.gmail.com>
References: <CANsKWwt4ORecf65OjEjyy1JOdgkszcJxUTO2H4tKYSDxAUK0+A@mail.gmail.com>
Message-ID: <8495F639-FA23-4F85-9637-80A9D6AC5EC5@umassmed.edu>

Hi Lahari,

Nitrogen starvation is a poor method of synchronization, especially for looking at S-phase progression, because release from the arrest is very heterogeneous.  cdc25 arrest and release works great (and cdc25-ts cell transform just fine). Alternatively, if you don't want to use mutant background, you might consider lactose-gradient synchronization <http://www.ncbi.nlm.nih.gov/pubmed/16498704>.  It is not as good as cdc25, but it is way better than nitrogen starvation.

Nick


On 10 Sep, 12, at 9:08 AM, lahari reddy wrote:

> Dear all,
>  
> I have to check the cell cycle profile of a deletion mutant (say gene related to S phase progession) that has been transformed with a suppressor gene. The suppressor transformed strains were starved of nitrogen (EMM-NH4Cl), but the FACS profile shows 15% of cells arrested in G1. The plasmid  carries a uracil marker, so I thought may be the nitrogen from uracil may hinder the arrest.
>  
> I would be pleased if you could clarify some of my doubts.
>  
> 1. Is the experimental approach discussed above can be done at all, because I didnt find evidences of articles discussing the FACS profile of a strain that has been transformed with an ectopic plamsid in s.pombe.(pardon me if didnt give a proper serach)
>  
> 2. I thought of alternative strategy where by my deletion strain has cdc25-22 background, but Im starting to worry whether transformation of this temerature sensitive strain works at all with lithium acetate protocol(growing @ 320C ) Even if plasmid can be transformed by electroporation, Whether arrest at higher temperature and  release at lower temperature affect the plasmid expression in  strains.
>  
>  
>                                                                                          Thank you
>  
>  
> Lahari Reddy
> Graduate student
> Institute of Life sciences
> Hyderabad
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist



From Erik.Boye at rr-research.no  Mon Sep 10 17:03:31 2012
From: Erik.Boye at rr-research.no (Erik Boye)
Date: Mon, 10 Sep 2012 18:03:31 +0200
Subject: [Pombelist] s.pombe cell cycle
In-Reply-To: <8495F639-FA23-4F85-9637-80A9D6AC5EC5@umassmed.edu>
References: <CANsKWwt4ORecf65OjEjyy1JOdgkszcJxUTO2H4tKYSDxAUK0+A@mail.gmail.com>
	<8495F639-FA23-4F85-9637-80A9D6AC5EC5@umassmed.edu>
Message-ID: <AFE5B2464F1EB547BBACE62CCE8E402001D111E5@mr3k6001.ad.medicalresearch.no>

Dear Lahari,
I am not sure I have understood your experiment - or that Nick did. It
seems to me that all you did was to starve your cells for nitrogen and
then look at the DNA distribution after awhile. And you were surprised
that only 15% of the cells had a 1C DNA content. It appears to me that
you did not use nitrogen starvation as a method to synchronize the
cells, but I am not sure. If this is what you want, the methods
suggested by Nick should be much better than starvation and release. 
Erik

-----Original Message-----
From: pombelist-bounces at sanger.ac.uk
[mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Rhind, Nick
Sent: 10. september 2012 17:55
To: lahari reddy
Cc: <pombelist at sanger.ac.uk>
Subject: Re: [Pombelist] s.pombe cell cycle

Hi Lahari,

Nitrogen starvation is a poor method of synchronization, especially for
looking at S-phase progression, because release from the arrest is very
heterogeneous.  cdc25 arrest and release works great (and cdc25-ts cell
transform just fine). Alternatively, if you don't want to use mutant
background, you might consider lactose-gradient synchronization
<http://www.ncbi.nlm.nih.gov/pubmed/16498704>.  It is not as good as
cdc25, but it is way better than nitrogen starvation.

Nick


On 10 Sep, 12, at 9:08 AM, lahari reddy wrote:

> Dear all,
>  
> I have to check the cell cycle profile of a deletion mutant (say gene
related to S phase progession) that has been transformed with a
suppressor gene. The suppressor transformed strains were starved of
nitrogen (EMM-NH4Cl), but the FACS profile shows 15% of cells arrested
in G1. The plasmid  carries a uracil marker, so I thought may be the
nitrogen from uracil may hinder the arrest.
>  
> I would be pleased if you could clarify some of my doubts.
>  
> 1. Is the experimental approach discussed above can be done at all,
because I didnt find evidences of articles discussing the FACS profile
of a strain that has been transformed with an ectopic plamsid in
s.pombe.(pardon me if didnt give a proper serach)
>  
> 2. I thought of alternative strategy where by my deletion strain has
cdc25-22 background, but Im starting to worry whether transformation of
this temerature sensitive strain works at all with lithium acetate
protocol(growing @ 320C ) Even if plasmid can be transformed by
electroporation, Whether arrest at higher temperature and  release at
lower temperature affect the plasmid expression in  strains.
>  
>  
>
Thank you
>  
>  
> Lahari Reddy
> Graduate student
> Institute of Life sciences
> Hyderabad
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist


_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://publists.sanger.ac.uk/mailman/listinfo/pombelist


From laharireddybiot at gmail.com  Tue Sep 11 12:54:07 2012
From: laharireddybiot at gmail.com (lahari reddy)
Date: Tue, 11 Sep 2012 17:24:07 +0530
Subject: [Pombelist] Pombelist Digest, Vol 5, Issue 2
In-Reply-To: <mailman.1.1347361202.23274.pombelist@sanger.ac.uk>
References: <mailman.1.1347361202.23274.pombelist@sanger.ac.uk>
Message-ID: <CANsKWwsfVC5-2ScL2BwQnnqoNUxFvPGmb54TvCFhJJac1CKOpA@mail.gmail.com>

Thanks one and all for suggestions..
Lahari Reddy

On Tue, Sep 11, 2012 at 4:30 PM, <pombelist-request at sanger.ac.uk> wrote:

> Send Pombelist mailing list submissions to
>         pombelist at sanger.ac.uk
>
> To subscribe or unsubscribe via the World Wide Web, visit
>         http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> or, via email, send a message with subject or body 'help' to
>         pombelist-request at sanger.ac.uk
>
> You can reach the person managing the list at
>         pombelist-owner at sanger.ac.uk
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Pombelist digest..."
>
>
> Today's Topics:
>
>    1. s.pombe cell cycle (lahari reddy)
>    2. Re: s.pombe cell cycle (Rhind, Nick)
>    3. Re: s.pombe cell cycle (Erik Boye)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Mon, 10 Sep 2012 18:38:53 +0530
> From: lahari reddy <laharireddybiot at gmail.com>
> Subject: [Pombelist] s.pombe cell cycle
> To: pombelist at sanger.ac.uk
> Message-ID:
>         <
> CANsKWwt4ORecf65OjEjyy1JOdgkszcJxUTO2H4tKYSDxAUK0+A at mail.gmail.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Dear all,
>
> I have to check the cell cycle profile of a deletion mutant (say gene
> related to S phase progession) that has been transformed with a suppressor
> gene. The suppressor transformed strains were starved of nitrogen
> (EMM-NH4Cl), but the FACS profile shows 15% of cells arrested in G1. The
> plasmid  carries a uracil marker, so I thought may be the nitrogen from
> uracil may hinder the arrest.
>
> I would be pleased if you could clarify some of my doubts.
>
> 1. Is the experimental approach discussed above can be done at all, because
> I didnt find evidences of articles discussing the FACS profile of a strain
> that has been transformed with an ectopic plamsid in s.pombe.(pardon me if
> didnt give a proper serach)
>
> 2. I thought of alternative strategy where by my deletion strain has
> cdc25-22 background, but Im starting to worry whether transformation of
> this temerature sensitive strain works at all with lithium acetate
> protocol(growing @ 320C ) Even if plasmid can be transformed by
> electroporation, Whether arrest at higher temperature and  release at lower
> temperature affect the plasmid expression in  strains.
>
>
>
> Thank you
>
>
> Lahari Reddy
> Graduate student
> Institute of Life sciences
> Hyderabad
> -------------- next part --------------
> An HTML attachment was scrubbed...
> URL: <
> http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120910/9b4419e5/attachment-0001.html
> >
>
> ------------------------------
>
> Message: 2
> Date: Mon, 10 Sep 2012 15:55:09 +0000
> From: "Rhind, Nick" <Nick.Rhind at umassmed.edu>
> Subject: Re: [Pombelist] s.pombe cell cycle
> To: lahari reddy <laharireddybiot at gmail.com>
> Cc: "<pombelist at sanger.ac.uk>" <pombelist at sanger.ac.uk>
> Message-ID: <8495F639-FA23-4F85-9637-80A9D6AC5EC5 at umassmed.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> Hi Lahari,
>
> Nitrogen starvation is a poor method of synchronization, especially for
> looking at S-phase progression, because release from the arrest is very
> heterogeneous.  cdc25 arrest and release works great (and cdc25-ts cell
> transform just fine). Alternatively, if you don't want to use mutant
> background, you might consider lactose-gradient synchronization <
> http://www.ncbi.nlm.nih.gov/pubmed/16498704>.  It is not as good as
> cdc25, but it is way better than nitrogen starvation.
>
> Nick
>
>
> On 10 Sep, 12, at 9:08 AM, lahari reddy wrote:
>
> > Dear all,
> >
> > I have to check the cell cycle profile of a deletion mutant (say gene
> related to S phase progession) that has been transformed with a suppressor
> gene. The suppressor transformed strains were starved of nitrogen
> (EMM-NH4Cl), but the FACS profile shows 15% of cells arrested in G1. The
> plasmid  carries a uracil marker, so I thought may be the nitrogen from
> uracil may hinder the arrest.
> >
> > I would be pleased if you could clarify some of my doubts.
> >
> > 1. Is the experimental approach discussed above can be done at all,
> because I didnt find evidences of articles discussing the FACS profile of a
> strain that has been transformed with an ectopic plamsid in s.pombe.(pardon
> me if didnt give a proper serach)
> >
> > 2. I thought of alternative strategy where by my deletion strain has
> cdc25-22 background, but Im starting to worry whether transformation of
> this temerature sensitive strain works at all with lithium acetate
> protocol(growing @ 320C ) Even if plasmid can be transformed by
> electroporation, Whether arrest at higher temperature and  release at lower
> temperature affect the plasmid expression in  strains.
> >
> >
> >
>                  Thank you
> >
> >
> > Lahari Reddy
> > Graduate student
> > Institute of Life sciences
> > Hyderabad
> > _______________________________________________
> > Pombelist mailing list
> > Pombelist at sanger.ac.uk
> > http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>
>
>
> ------------------------------
>
> Message: 3
> Date: Mon, 10 Sep 2012 18:03:31 +0200
> From: "Erik Boye" <Erik.Boye at rr-research.no>
> Subject: Re: [Pombelist] s.pombe cell cycle
> To: "Rhind, Nick" <Nick.Rhind at umassmed.edu>,    "lahari reddy"
>         <laharireddybiot at gmail.com>
> Cc: pombelist at sanger.ac.uk
> Message-ID:
>         <
> AFE5B2464F1EB547BBACE62CCE8E402001D111E5 at mr3k6001.ad.medicalresearch.no>
>
> Content-Type: text/plain;       charset="us-ascii"
>
> Dear Lahari,
> I am not sure I have understood your experiment - or that Nick did. It
> seems to me that all you did was to starve your cells for nitrogen and
> then look at the DNA distribution after awhile. And you were surprised
> that only 15% of the cells had a 1C DNA content. It appears to me that
> you did not use nitrogen starvation as a method to synchronize the
> cells, but I am not sure. If this is what you want, the methods
> suggested by Nick should be much better than starvation and release.
> Erik
>
> -----Original Message-----
> From: pombelist-bounces at sanger.ac.uk
> [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Rhind, Nick
> Sent: 10. september 2012 17:55
> To: lahari reddy
> Cc: <pombelist at sanger.ac.uk>
> Subject: Re: [Pombelist] s.pombe cell cycle
>
> Hi Lahari,
>
> Nitrogen starvation is a poor method of synchronization, especially for
> looking at S-phase progression, because release from the arrest is very
> heterogeneous.  cdc25 arrest and release works great (and cdc25-ts cell
> transform just fine). Alternatively, if you don't want to use mutant
> background, you might consider lactose-gradient synchronization
> <http://www.ncbi.nlm.nih.gov/pubmed/16498704>.  It is not as good as
> cdc25, but it is way better than nitrogen starvation.
>
> Nick
>
>
> On 10 Sep, 12, at 9:08 AM, lahari reddy wrote:
>
> > Dear all,
> >
> > I have to check the cell cycle profile of a deletion mutant (say gene
> related to S phase progession) that has been transformed with a
> suppressor gene. The suppressor transformed strains were starved of
> nitrogen (EMM-NH4Cl), but the FACS profile shows 15% of cells arrested
> in G1. The plasmid  carries a uracil marker, so I thought may be the
> nitrogen from uracil may hinder the arrest.
> >
> > I would be pleased if you could clarify some of my doubts.
> >
> > 1. Is the experimental approach discussed above can be done at all,
> because I didnt find evidences of articles discussing the FACS profile
> of a strain that has been transformed with an ectopic plamsid in
> s.pombe.(pardon me if didnt give a proper serach)
> >
> > 2. I thought of alternative strategy where by my deletion strain has
> cdc25-22 background, but Im starting to worry whether transformation of
> this temerature sensitive strain works at all with lithium acetate
> protocol(growing @ 320C ) Even if plasmid can be transformed by
> electroporation, Whether arrest at higher temperature and  release at
> lower temperature affect the plasmid expression in  strains.
> >
> >
> >
> Thank you
> >
> >
> > Lahari Reddy
> > Graduate student
> > Institute of Life sciences
> > Hyderabad
> > _______________________________________________
> > Pombelist mailing list
> > Pombelist at sanger.ac.uk
> > http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>
>
> ------------------------------
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
> End of Pombelist Digest, Vol 5, Issue 2
> ***************************************
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120911/409d173a/attachment.htm>

From j.bahler at ucl.ac.uk  Wed Sep 12 20:11:04 2012
From: j.bahler at ucl.ac.uk (Bahler, Jurg)
Date: Wed, 12 Sep 2012 19:11:04 +0000
Subject: [Pombelist] pombe 2013: 24-29 June 2013
Message-ID: <74249C6FD1C7CB4CB0E1451F04F8D19432169342@DB3PRD0104MB132.eurprd01.prod.exchangelabs.com>

Dear Friends & Colleagues,

This is just to remind everybody that the 7th International Fission Yeast Meeting (pombe2013) will take place in London (UK), starting on Monday 24th June and ending on Saturday 29th June 2013.
They called the London 2012 Olympics & Paralympics "the greatest show on earth", but of course they haven't seen pombe 2013 yet...

Jacky and I look forward to welcoming you in London next year. Please mark your diaries and stay tuned for more details.

All the best,
-J?rg

---
J?rg B?hler
Professor of Systems Biology

University College London
Department of Genetics, Evolution & Environment
and UCL Cancer Institute
Darwin Building, Gower Street
London WC1E 6BT
U.K.

P: +44(0)20 3108 1602 (Internal x51602)
F: +44(0)20 7679 7096
E: j.bahler at ucl.ac.uk<mailto:j.bahler at ucl.ac.uk>
W: www.bahlerlab.info<http://www.sanger.ac.uk/PostGenomics/S_pombe/>

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120912/abd12f8c/attachment.htm>

From jacqueline.hayles at cancer.org.uk  Mon Sep 17 11:10:59 2012
From: jacqueline.hayles at cancer.org.uk (Jacqueline Hayles)
Date: Mon, 17 Sep 2012 11:10:59 +0100
Subject: [Pombelist] Pombe Club
Message-ID: <CC7CB5C3.F2A1%jacqueline.hayles@cancer.org.uk>

I have had a flurry of emails asking if it is OK still to come along to the Pombe Club tomorrow even if they had not previously sent an email saying they were attending.  Just to let you know it is fine and everyone is welcome.

Regards

Jacky

Jacqueline Hayles
Cell Cycle Laboratory
Cancer Research UK, London Research Institute
44 Lincoln's Inn Fields
London WC2A 3LY, UNITED KINGDOM

Tel ::: +44 (0) 20 7269 3105
Fax ::: +44 (0) 20 7269 3258
email   jacqueline.hayles at cancer.org.uk

[cid:6A256EA4-3032-44C8-9130-9E5F0277F4AA]




NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103)
A company limited by guarantee.  Registered company in England and Wales (4325234) and the Isle of Man (5713F).
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120917/6bea221d/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 8E841165-810C-4AE2-9EEE-1E45A81D22C2.png
Type: image/png
Size: 61902 bytes
Desc: 8E841165-810C-4AE2-9EEE-1E45A81D22C2.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120917/6bea221d/attachment-0001.png>

From byg2013 at nottingham.ac.uk  Wed Sep 19 19:19:36 2012
From: byg2013 at nottingham.ac.uk (Pl-Byg2013)
Date: Wed, 19 Sep 2012 19:19:36 +0100
Subject: [Pombelist] British Yeast Group Meeting 2013
Message-ID: <CC7FCB4A.5A13%PL-byg2013@exmail.nottingham.ac.uk>

An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120919/a86f5d56/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: BYG2013_poster.pdf
Type: application/pdf
Size: 520142 bytes
Desc: BYG2013_poster.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120919/a86f5d56/attachment-0001.pdf>

From byg2013 at nottingham.ac.uk  Thu Sep 20 13:53:17 2012
From: byg2013 at nottingham.ac.uk (Pl-Byg2013)
Date: Thu, 20 Sep 2012 13:53:17 +0100
Subject: [Pombelist] British Yeast Group meeting 2013
Message-ID: <CC80D04F.5A56%PL-byg2013@exmail.nottingham.ac.uk>

An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120920/8e1b56b4/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: BYG2013_poster.pdf
Type: application/pdf
Size: 508162 bytes
Desc: BYG2013_poster.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20120920/8e1b56b4/attachment-0001.pdf>

From rachel.brown.10 at ucl.ac.uk  Mon Oct  1 10:50:35 2012
From: rachel.brown.10 at ucl.ac.uk (Rachel Brown)
Date: Mon, 1 Oct 2012 10:50:35 +0100
Subject: [Pombelist] Transposons in S.pombe
Message-ID: <CAJWXR7rtotHZw=WEN83qkNz_4cxwKKfqPPrz3=vsMdQ_JVA71w@mail.gmail.com>

I am a new PhD student at University College London and plan to perform
random mutagenesis screens in *S.pombe* using transposons.

Two transposon-based systems have been described for *S.pombe* (references
below) but very little has been published about them (in *S.pombe*).

I was therefore wondering if anyone has any experience with the groups
below or the transposon system that they might be able to provide me? If
anyone has any advice etc, it would be greatly appreciated!

Thank you,

Rachel

1. Evertts et al (2007). *The hermes transposon **of Musca domestica is an
efficient tool for the mutagenesis of Schizosaccharomyces pombe. **Genetics.
*177(4):2519-2523.


2. Li et al. (2011). *A piggyBac transposon-based mutagenesis system for
the fission yeast Schizossaccharomyces pombe.* *Nucleic Acids Research. *
39(6).* *

--
Rachel Brown BSc (Hons), MSc.
PhD Researcher
MRC Laboratory for Molecular Cell Biology
University College London
Gower Street
London
WC1E 6BY
UK

rachel.brown.10 at ucl.ac.uk
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121001/f4167785/attachment.htm>

From mstyers at bsc.edu  Mon Oct  1 17:02:56 2012
From: mstyers at bsc.edu (Styers, Melanie L.)
Date: Mon, 1 Oct 2012 16:02:56 +0000
Subject: [Pombelist] Southeast Regional Yeast Meeting 2013
Message-ID: <7FA1D710DC9A804BA0343417D58474A13D67578D@BL2PRD0611MB423.namprd06.prod.outlook.com>

Hello,



I just wanted to pass along some information about an upcoming meeting to those of you in the Southeastern US.

The 20th annual Southeast Regional Yeast Meeting (SERYM) will be held March 8-10, 2013 at the University of Alabama at Birmingham (UAB) in Birmingham, AL. For the 20th annual meeting, our keynote speaker is Randy Schekman, a pioneer in characterizing the mechanisms of membrane transport from yeast to humans.
The registration site will soon be open. For now, please mark this weekend on your calendar, you won?t want to miss it!

Thank you,
SERYM 2013 organizers

Pamela Hansen, BSC
Marla Hertz, UAB
David Schneider, UAB
Melanie Styers, BSC
Robert van Waardenburg, UAB
__________________________________________
Melanie L. Styers, Ph.D.
Assistant Professor of Biology
Birmingham-Southern College
Box 549022
Birmingham, AL  35254
SSC 245
Office:  205.226.4882
Fax:  205.226.3078
Email:  mstyers at bsc.edu<mailto:mstyers at bsc.edu>

Explore...Birmingham-Southern College
www.bsc.edu<http://www.bsc.edu/>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121001/ffc8f7b5/attachment.htm>

From paul.nurse at royalsociety.org  Mon Oct  1 17:10:45 2012
From: paul.nurse at royalsociety.org (Nurse, Paul)
Date: Mon, 1 Oct 2012 17:10:45 +0100
Subject: [Pombelist] Postdoc position
Message-ID: <B6BC0CFD4EA8FF4DBF3DE91E99DE731E011237955164@edwardiii.rsnet.local>

Dear All,

Tarun Kapoor (Rockefeller University) and I are collaborating on a project that involves using S. Pombe for chemical biology. We have an open postdoctoral position in the Rockefeller University, New York and are looking for a candidate who is independent, motivated, and has experience working with the fission yeast. The paper in Chemistry and Biology (y2012, v19, p893) should help explain the types of projects we are planning.

This position will be funded for one year in the first instance.
Applications should be sent to my Lab Manager at the Rockefeller University, before November 15, 2012. 

Lab Manager: Ryoko Mandeville: mandevr at rockefeller.edu

Thanks,
Paul Nurse


*************************************************************************
This email is sent on behalf of The Royal Society, 6-9 Carlton House Terrace, London SW1Y 5AG, United Kingdom. 

You should carry out your own virus check before opening any attachment. The Royal Society accepts no liability for any loss or damage which may be caused by software viruses or interception or interruption of this email.
The contents of this email and any attachments are intended for the confidential use of the named recipient(s) only. They may be legally privileged and should not be communicated to or relied upon by any person without our express written consent. If you are not an addressee (or you have received this mail in error) please notify us immediately by email to: ithelpdesk at royalsociety.org

Registered charity no. 207043
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121001/c9601b29/attachment.htm>

From jacqueline.hayles at cancer.org.uk  Tue Oct  2 10:24:01 2012
From: jacqueline.hayles at cancer.org.uk (Jacqueline Hayles)
Date: Tue, 2 Oct 2012 10:24:01 +0100
Subject: [Pombelist] Next Pombe Club date
Message-ID: <CC907141.F859%jacqueline.hayles@cancer.org.uk>


Hi Everyone

 The next Pombe Club will be on January 28th 2013 at the LRI (5-8pm).  I'm will send the programme later, this just to let you know the date.

Regards

Jacky

Jacqueline Hayles
Cell Cycle Laboratory
Cancer Research UK, London Research Institute
44 Lincoln's Inn Fields
London WC2A 3LY, UNITED KINGDOM

Tel ::: +44 (0) 20 7269 3105
Fax ::: +44 (0) 20 7269 3258
email   jacqueline.hayles at cancer.org.uk

[cid:17EF367D-A64C-49E3-9840-674A02CF34D2]



NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103)
A company limited by guarantee.  Registered company in England and Wales (4325234) and the Isle of Man (5713F).
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121002/49021be1/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: AB2C9924-2171-4F47-8BF1-A7B365D677A5.png
Type: image/png
Size: 54826 bytes
Desc: AB2C9924-2171-4F47-8BF1-A7B365D677A5.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121002/49021be1/attachment-0001.png>

From huberman at buffalo.edu  Tue Oct  2 18:03:38 2012
From: huberman at buffalo.edu (Joel Huberman)
Date: Tue, 2 Oct 2012 13:03:38 -0400
Subject: [Pombelist] Instructional videos for freeze-grinding fission yeast
	cells
Message-ID: <47345685-6918-4DFD-A416-B2D98045AF20@buffalo.edu>

Dear PombeList,

	My colleague, Dr. Robert Givens, and I are making publicly available some instructional videos showing (i) how to freeze-grind fission yeast cells in a precision-controlled motorized mortar-grinder under liquid nitrogen, (ii) how to isolate high-molecular-weight DNA from the liberated nuclei, and (iii) how to prepare powdered, frozen buffer for purposes of rapid, uniform equilibration with frozen cell grindate. All three of these videos are based on techniques described in detail in our 2011 publication, and utilized in our 2012 publication:

Givens RM, Mesner LD, Hamlin JL, Buck MJ, Huberman JA. Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen. BMC Research Notes 4:499, 2011.

Givens RM, Lai WKM, Rizzo JM, Bard JE, Mieczkowski PA, Leatherwood J, Huberman JA, Buck MJ. Chromatin architectures at fission yeast transcriptional promoters and replication origins. Nucleic Acids Research 40: 7176-7189, 2012.

	The videos can be downloaded from my web site, http://joelhuberman.net/FreezeGrindMovies/index.html. They can also be accessed from YouTube:

Preparation of high-molecular-weight DNA: http://youtu.be/GMx05Thqx_A
Fine Freeze-Grinding of Buffer: http://youtu.be/0yccOXJWlIo
Freeze-Grinding Fission Yeast Cells: http://youtu.be/7bW19QmaZY0

	We hope these videos will prove useful to you. If any questions arise, you can contact me (huberman at buffalo.edu) or Dr. Givens (ientist at verizon.net).


*********************************
Dr. Joel A. Huberman
Emeritus Professor
Department of Molecular & Cellular Biology
Roswell Park Cancer Institute
Elm & Carlton Streets
Buffalo, NY  14263-0001
(716) 845-3047  Phone
huberman at buffalo.edu
*********************************




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121002/a857c475/attachment.htm>

From hoffmacs at bc.edu  Thu Oct  4 15:18:13 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Thu, 4 Oct 2012 10:18:13 -0400
Subject: [Pombelist] Why doesn't alpha-aminoadipate work in pombe?
Message-ID: <17ED0EA0-8E9D-4AE7-BA20-DB39C226303D@bc.edu>

I was thinking about counterselection and was wondering if any can explain why wild type S. cerevisiae is sensitive to alpha-aminoadipate, but S. pombe is resistant?  I think I put Sc LYS2 in pombe once, but it did not become sensitive (then again, I am not sure that it was expressed).

Cheers,

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"





From FVerde at med.miami.edu  Tue Oct  9 18:41:01 2012
From: FVerde at med.miami.edu (Verde, Fulvia)
Date: Tue, 9 Oct 2012 13:41:01 -0400
Subject: [Pombelist] Postdoctoral position available
Message-ID: <CC99CDC3.4939A%fverde@med.miami.edu>

Dear all,

A Postdoctoral position is available in my laboratory to study the process of spatial self-organization in fission yeast and in particular the regulation of Cdc42 GTPase dynamics. For background information on this project, please see our recent paper ?Oscillatory dynamics of Cdc42 GTPase in the control of polarized growth? in Science. (2012, 337(6091):239-43). I am looking for motivated candidates that ideally have some experience with molecular and/or microscopy approaches in fission or budding yeast.

The position is initially funded for two years.

Best wishes Fulvia Verde



Fulvia Verde, Ph.D.
Associate Professor
Molecular and Cellular Pharmacology
University of Miami, Miller School of Medicine

Phone:   305-243-3106 (office)
    305-243-3643 (lab)
Fax:     305-243-4555

email: fverde at med.miami.edu

URL: www.biomed.miami.edu/verde

Mailing address:
Department of Molecular and Cellular Pharmacology (R-189)
University of Miami, Miller School of Medicine
P.O. Box 016189
Miami, FL 33101

Packages and lab location:
Department of Molecular and Cellular Pharmacology
University of Miami, Miller School of Medicine
1600 NW 10th Avenue, RMSB 6165 (R-189)
Miami, FL 33136


From daniel.lew at duke.edu  Wed Oct 10 02:11:42 2012
From: daniel.lew at duke.edu (Daniel Lew, Ph.D.)
Date: Wed, 10 Oct 2012 01:11:42 +0000
Subject: [Pombelist] Save the date: 2013 Cold Spring Harbor Yeast Cell
 Biology Meeting in November
Message-ID: <CC9A438C.F215%daniel.lew@dm.duke.edu>

Fellow yeast cell biologists:

We wanted to alert you that the 2013 Cold Spring Harbor Yeast Cell Biology meeting, traditionally held in August, has been moved to November 5-9 of next year.  After a strong showing from S. pombe researchers in 2011, we hope that the later date will allow more S. pombe cell biologists to join us in 2013.  We hope to see you there!

Ken Sawin,  Martha Cyert, and Daniel Lew
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121010/793c2c43/attachment.htm>

From damien.coudreuse at univ-rennes1.fr  Sun Oct 14 19:11:16 2012
From: damien.coudreuse at univ-rennes1.fr (Damien_IGDR)
Date: Sun, 14 Oct 2012 20:11:16 +0200
Subject: [Pombelist] Post-doctoral positions funded by the ERC
Message-ID: <E50CDA9C-C983-42BA-A65C-6A58861E4306@univ-rennes1.fr>

Dear all,

Two post-doctoral positions funded by an ERC Starting Grant will be available starting in 2013 in my laboratory at the Institute of Genetics and Development in Rennes, France (see attached PDF). Our group investigates different aspects of cell cycle regulation and evolution using a synthetic biology approach in fission yeast. I would appreciate it if you could pass on this information to anyone who you think may be interested.

Thank you very much for your help.


Best wishes,

Damien


**********************************************
Damien Coudreuse, Group Leader
Team SyntheCell
Institute of Genetics and Development
CNRS UMR 6290
2, Avenue du Pr. L?on Bernard
35043 Rennes Cedex
FRANCE
Phone: +33 (0)2 23 23 44 37








-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121014/3d48e6d5/attachment-0002.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: PostdocERC.pdf
Type: application/pdf
Size: 120308 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121014/3d48e6d5/attachment-0001.pdf>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121014/3d48e6d5/attachment-0003.htm>

From mdas at utk.edu  Mon Oct 15 00:20:10 2012
From: mdas at utk.edu (Das, Maitreyi)
Date: Sun, 14 Oct 2012 23:20:10 +0000
Subject: [Pombelist] post doctoral position
Message-ID: <EEB50CC86418F347BE5A5182859EE9B3324A8C41@kmbx4.utk.tennessee.edu>

Dear All



A post-doctoral  position is available in my lab at the University of Tennessee at Knoxville. Our lab studies cell polarity and growth control in fission yeast using live-cell imaging, molecular biology and genetics approaches. For further details please see the attached advertisement. Thank you.



Regards

Maitreyi



Maitreyi Das, PhD

Assistant Professor, BCMB

University of Tennessee, Knoxville

F235, Walters Life Sciences

1414 Cumberland Avenue

Knoxville, TN 37996


-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121014/e6a9485c/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Das Post Doc.pdf
Type: application/pdf
Size: 390604 bytes
Desc: Das Post Doc.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121014/e6a9485c/attachment-0001.pdf>

From forsburg at usc.edu  Mon Oct 15 19:10:50 2012
From: forsburg at usc.edu (SLForsburg)
Date: Mon, 15 Oct 2012 11:10:50 -0700
Subject: [Pombelist] ChIP seq protocol
Message-ID: <D8619F82-1ED2-44F9-B5EA-65516CE68B33@usc.edu>

Hi folks,
We are looking for ChIP seq protocols.  Can you help?  Also would like an idea of how many reads/sample and samples/lane you are running these days.

Thanks!

Susan


 
{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 






From ozan.aygun at nih.gov  Mon Oct 15 22:26:57 2012
From: ozan.aygun at nih.gov (Aygun, Ozan (NIH/NCI) [F])
Date: Mon, 15 Oct 2012 17:26:57 -0400
Subject: [Pombelist] Cell cycle analysis of morphologically abnormal S.pombe
 mutants by FACS
In-Reply-To: <D8619F82-1ED2-44F9-B5EA-65516CE68B33@usc.edu>
Message-ID: <CCA1F5AF.44BE%ozan.aygun@nih.gov>

Dear all,

I am trying to find out whether there is a method of analyzing DNA content (N or 2N) of morphologically abnormal (such as extremely elongated) S.pombe mutants by FACS? I have been trying different combinations of forward and side scatter detectors but don't have much success so far. Any suggestions or advice you may have will be extremely helpful.

Sincerely,

Ozan

P.S: I use sytox green to stain DNA instead of PI.

***************************************
Ozan Aygun, Ph.D.

Laboratory of Biochemistry and Molecular Biology
National Cancer Institute
National Institutes of Health
Bethesda, USA

E-mail: ayguno at mail.nih.gov<mailto:ayguno at mail.nih.gov>
**************************************




From chris.norbury at path.ox.ac.uk  Mon Oct 15 22:46:33 2012
From: chris.norbury at path.ox.ac.uk (Chris Norbury)
Date: Mon, 15 Oct 2012 22:46:33 +0100
Subject: [Pombelist] Cell cycle analysis of morphologically abnormal
	S.pombe mutants by FACS
In-Reply-To: <CCA1F5AF.44BE%ozan.aygun@nih.gov>
References: <CCA1F5AF.44BE%ozan.aygun@nih.gov>
Message-ID: <0758D24D-7C35-4223-AF61-2AEF2628F1FA@path.ox.ac.uk>

Hi Ozan,

With PI staining, I've used red fluorescence against FSC as a bivariate contour plot, which should be pretty unambiguous, even with very elongated cells; they will appear as inclined sausages, with origins clearly at the position of 1N or 2N reference cultures!

Hope this helps.

All the best,

Chris

On 15 Oct 2012, at 22:26, "Aygun, Ozan (NIH/NCI) [F]" <ozan.aygun at nih.gov> wrote:

> Dear all,
> 
> I am trying to find out whether there is a method of analyzing DNA content (N or 2N) of morphologically abnormal (such as extremely elongated) S.pombe mutants by FACS? I have been trying different combinations of forward and side scatter detectors but don't have much success so far. Any suggestions or advice you may have will be extremely helpful.
> 
> Sincerely,
> 
> Ozan
> 
> P.S: I use sytox green to stain DNA instead of PI.
> 
> ***************************************
> Ozan Aygun, Ph.D.
> 
> Laboratory of Biochemistry and Molecular Biology
> National Cancer Institute
> National Institutes of Health
> Bethesda, USA
> 
> E-mail: ayguno at mail.nih.gov<mailto:ayguno at mail.nih.gov>
> **************************************
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist



From Nick.Rhind at umassmed.edu  Mon Oct 15 23:02:37 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Mon, 15 Oct 2012 22:02:37 +0000
Subject: [Pombelist] Cell cycle analysis of morphologically abnormal
 S.pombe mutants by FACS
In-Reply-To: <CCA1F5AF.44BE%ozan.aygun@nih.gov>
References: <CCA1F5AF.44BE%ozan.aygun@nih.gov>
Message-ID: <C58B901E-3C7A-461E-A353-2AA77C7A3FE9@umassmed.edu>

Hi Ozan,

We isolate the nuclei from cells before FACS to avoid morphology issues.  The protocol in the attached methods paper (page 176) is easy (zymolyase treat and sonicate the ethanol-fixxed cells to release the nuclei) and gives beautiful FACS from ugly cells.

Nick


On 15 Oct, 12, at 5:26 PM, Aygun, Ozan (NIH/NCI) [F] wrote:

> Dear all,
> 
> I am trying to find out whether there is a method of analyzing DNA content (N or 2N) of morphologically abnormal (such as extremely elongated) S.pombe mutants by FACS? I have been trying different combinations of forward and side scatter detectors but don't have much success so far. Any suggestions or advice you may have will be extremely helpful.
> 
> Sincerely,
> 
> Ozan
> 
> P.S: I use sytox green to stain DNA instead of PI.
> 
> ***************************************
> Ozan Aygun, Ph.D.
> 
> Laboratory of Biochemistry and Molecular Biology
> National Cancer Institute
> National Institutes of Health
> Bethesda, USA
> 
> E-mail: ayguno at mail.nih.gov<mailto:ayguno at mail.nih.gov>
> **************************************
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist


-------------- next part --------------
A non-text attachment was scrubbed...
Name: Forsburg and Rhind (2006) Yeast.pdf
Type: application/pdf
Size: 135720 bytes
Desc: Forsburg and Rhind (2006) Yeast.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121015/dff8d454/attachment-0001.pdf>

From kristin.scott at duke.edu  Tue Oct 16 20:47:32 2012
From: kristin.scott at duke.edu (Kristin C Scott, Ph.D.)
Date: Tue, 16 Oct 2012 19:47:32 +0000
Subject: [Pombelist] unintentional non-sporulating diploid
Message-ID: <B88438A280491146BB0DF15AAB5904E6015FB6B9@ex-mbg-06.win.duke.edu>

Dear All,

This is our first try at making diploids, so please bear with my inexperience.  I intended to make a heterozygous sporulating diploid as a way to monitor chromosome loss upon the breakdown of the diploid.  Using ade6 complementing alleles, the phloxin test for distinguishing diploids from haploids, I selected single colonies, resuspended at a low concentration and plated non-selectively (with the intent of replica plating for markers).

However, after plating on low adenine plates, most colonies remained white and under a microscope all colonies (both white and pinks?!) remain diploid [longer, straighter asci and some have more than 4 spores].  Even after several days on EMM without selection.  I will do the iodine stain test to confirm lack of sporulation.

I have not tinkered with any mating or sporulation genes in these strains, and the mating type locus in parent cells is wild-type (one h+, one h-), so I assumed that the diploid would break down immediately.

The haploid mutant parent strain is viable, though they do grow poorly.  Does lack of sporulation in this context suggest that our mutation, though not absolutely necessary for viability, enhances viability?  Will they break down eventually and/or is there a way to speed breakdown up, perhaps with glusulase?

Please advise if there are other "standard" things that we should be doing that I am not aware of,  primary literature that I should be reading, and/or suggestions for follow-up experiments.

Many thanks,
Kristin




Kristin C. Scott, Ph.D.
Duke Institute for Genome Sciences and Policy
Duke University
100 Science Drive, Room 2345
Durham, NC 27008

919-684-7938 (office)
919-684-2434 (lab)



-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121016/ee40b1b7/attachment.htm>

From forsburg at usc.edu  Tue Oct 16 22:10:58 2012
From: forsburg at usc.edu (SLForsburg)
Date: Tue, 16 Oct 2012 14:10:58 -0700
Subject: [Pombelist] unintentional non-sporulating diploid
In-Reply-To: <B88438A280491146BB0DF15AAB5904E6015FB6B9@ex-mbg-06.win.duke.edu>
References: <B88438A280491146BB0DF15AAB5904E6015FB6B9@ex-mbg-06.win.duke.edu>
Message-ID: <D7578670-4736-41AB-A853-B41338BB503F@usc.edu>

IT's useful to remember that (unlike cerevisiae) pombe will sporulate at the sniff of a loss of nitrogen;  therefore,  culture on minimal media enhances selection of non-sporulating mutants.  It's essential to use YES as much as possible and confrim as you go along.  Having a wild type cross alongside your mutant may help.

"Breakdown" only occurs once they have sporulated.  If what you have is just unsporulated diploids, glusalase will simply kill them.

Some suggestions on how to make diploids are here:

http://www-bcf.usc.edu/~forsburg/diploids.html


On Oct 16, 2012, at 12:47 PM, Kristin C Scott, Ph.D. wrote:

> Dear All,
> 
> This is our first try at making diploids, so please bear with my inexperience.  I intended to make a heterozygous sporulating diploid as a way to monitor chromosome loss upon the breakdown of the diploid.  Using ade6 complementing alleles, the phloxin test for distinguishing diploids from haploids, I selected single colonies, resuspended at a low concentration and plated non-selectively (with the intent of replica plating for markers).  
> 
> However, after plating on low adenine plates, most colonies remained white and under a microscope all colonies (both white and pinks?!) remain diploid [longer, straighter asci and some have more than 4 spores].  Even after several days on EMM without selection.  I will do the iodine stain test to confirm lack of sporulation.  
> 
> I have not tinkered with any mating or sporulation genes in these strains, and the mating type locus in parent cells is wild-type (one h+, one h-), so I assumed that the diploid would break down immediately.
> 
> The haploid mutant parent strain is viable, though they do grow poorly.  Does lack of sporulation in this context suggest that our mutation, though not absolutely necessary for viability, enhances viability?  Will they break down eventually and/or is there a way to speed breakdown up, perhaps with glusulase? 
> 
> Please advise if there are other "standard" things that we should be doing that I am not aware of,  primary literature that I should be reading, and/or suggestions for follow-up experiments. 
> 
> Many thanks,
> Kristin 
> 
> 
> 
> 
> Kristin C. Scott, Ph.D.
> Duke Institute for Genome Sciences and Policy
> Duke University
> 100 Science Drive, Room 2345
> Durham, NC 27008
> 
> 919-684-7938 (office)
> 919-684-2434 (lab)
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist

 
{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121016/095a3667/attachment.htm>

From naomi.barrett.11 at ucl.ac.uk  Mon Oct 22 12:06:28 2012
From: naomi.barrett.11 at ucl.ac.uk (Barrett, Naomi)
Date: Mon, 22 Oct 2012 11:06:28 +0000
Subject: [Pombelist] demonstration for ONIX microfluidics system
Message-ID: <B855C49A9FB44740A168AEC6990EB3BA7A895ADF@DB3PRD0104MB156.eurprd01.prod.exchangelabs.com>

Dear all,

We are looking into buying an ONIX Microfluidic perfusion platform for live cell imaging. Does anyone have experience using this system and can recommend it?

Additionally, we are having a demonstration shortly, does anyone have any suggestions as to the type of microscope that we should be using with the ONIX system?

Many thanks,

Naomi Barrett


Department of Genetics, Evolution and Environment
University College London
Darwin Building, Gower Street,
WC1E 6BT, London,UK

E-mail: naomi.barrett.11 at ucl.ac.uk
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121022/1a3cd93c/attachment.htm>

From hoffmacs at bc.edu  Tue Oct 23 02:35:58 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Mon, 22 Oct 2012 21:35:58 -0400
Subject: [Pombelist] RNA viruses in S. pombe?
Message-ID: <BEE2DE3B-0939-490A-879C-02F9DDBFF74F@bc.edu>

Hi,

I was wondering if anyone knows of evidence of RNA viruses in S. pombe, such as the totiviruses in S. cerevisiae.  Have any RNA-seq projects found them? Thanks.

Cheers,

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"





From jilong.liu at dpag.ox.ac.uk  Tue Oct 23 11:34:32 2012
From: jilong.liu at dpag.ox.ac.uk (Ji-Long Liu)
Date: Tue, 23 Oct 2012 10:34:32 +0000
Subject: [Pombelist] postdoc position in Oxford
In-Reply-To: <EEB50CC86418F347BE5A5182859EE9B3324A8C41@kmbx4.utk.tennessee.edu>
References: <EEB50CC86418F347BE5A5182859EE9B3324A8C41@kmbx4.utk.tennessee.edu>
Message-ID: <084452d8-7282-4300-90df-041545210133@HUB02.ad.oak.ox.ac.uk>

Dear All

A postdoc position (MRC Career Development Fellowship) is available in my lab at the MRC Functional Genomics Unit, University of Oxford. We are interested in metabolic compartmentation using S. pombe or Drosophila as model systems. For further info please see below a link to our advert at Naturejobs. Many thanks.

http://www.nature.com/naturejobs/science/jobs/288640-MRC-Career-Development-Fellowship

Best wishes

Jilong

Ji-Long Liu, PhD
Programme Leader, MRC Functional Genomics Unit
Department of Physiology, Anatomy and Genetics
University of Oxford
South Parks Road, Oxford OX1 3PT
Tel: (01865) 285833          E-mail: jilong.liu at dpag.ox.ac.uk
http://groups.mrcfgu.ox.ac.uk/liu-group
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121023/858a7e19/attachment.htm>

From Nick.Rhind at umassmed.edu  Tue Oct 23 13:59:50 2012
From: Nick.Rhind at umassmed.edu (Rhind, Nick)
Date: Tue, 23 Oct 2012 12:59:50 +0000
Subject: [Pombelist] RNA viruses in S. pombe?
In-Reply-To: <BEE2DE3B-0939-490A-879C-02F9DDBFF74F@bc.edu>
References: <BEE2DE3B-0939-490A-879C-02F9DDBFF74F@bc.edu>
Message-ID: <EABBE3C9-C075-4F41-B8A3-09CB64EE3466@umassmed.edu>

Hi Charlie,

Nothing obvious came out of the Schizo RNA-seq project at the Broad.  The transcriptomes were assembled independently of the genomes, so we should have found viral sequences, if they were there.  However, nobody looked specifically.  The data is all available for anyone who would like to look.

Nick

On Oct 22, 2012, at 9:36 PM, "Charlie Hoffman" <hoffmacs at bc.edu> wrote:

> Hi,
> 
> I was wondering if anyone knows of evidence of RNA viruses in S. pombe, such as the totiviruses in S. cerevisiae.  Have any RNA-seq projects found them? Thanks.
> 
> Cheers,
> 
> Charlie Hoffman
> hoffmacs at bc.edu
> "I'd rather be fission"
> 
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist


From jacqueline.hayles at cancer.org.uk  Fri Oct 26 12:52:41 2012
From: jacqueline.hayles at cancer.org.uk (Jacqueline Hayles)
Date: Fri, 26 Oct 2012 12:52:41 +0100
Subject: [Pombelist] Pombe Club
Message-ID: <CCB03819.36B0%jacqueline.hayles@cancer.org.uk>

Dear All

Please find attached the programme for next Pombe Club on January 28th 2013

Regards

Jacky

Jacqueline Hayles
Cell Cycle Laboratory
Cancer Research UK
London Research Institute
44, Lincoln's Inn Fields
London
WC2A 3LY
UK
Tel +44 207 269 3105
Email jacqueline.hayles at cancer.org.uk

[cid:4C41CB18-3259-46E3-A066-56EE8286EEE8]

NOTICE AND DISCLAIMER
This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. 

We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. 
Cancer Research UK
Registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103)
A company limited by guarantee.  Registered company in England and Wales (4325234) and the Isle of Man (5713F).
Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121026/98075268/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: 429B74C6-7439-48E3-8D51-CCABA709AB49.png
Type: image/png
Size: 56718 bytes
Desc: 429B74C6-7439-48E3-8D51-CCABA709AB49.png
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121026/98075268/attachment-0001.png>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Pombe Club Notice 28.01.13 .doc
Type: application/msword
Size: 205312 bytes
Desc: Pombe Club Notice 28.01.13 .doc
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121026/98075268/attachment-0001.doc>

From Sarah.Lambert at curie.fr  Wed Oct 31 16:48:17 2012
From: Sarah.Lambert at curie.fr (Lambert Sarah)
Date: Wed, 31 Oct 2012 16:48:17 +0000
Subject: [Pombelist] Post-doc position Lambert's lab.
Message-ID: <B68D2365E185A6469F4DCD245E9D5BF112F62072@mbxparis02.recherche.curie.fr>

Dear all,

A post-doctoral position will be available starting in 2013 in my laboratory at the Institut Curie, in Orsay, France (see attached PDF). My group investigates interplays between chromatin assembly and DNA repair pathways in fission yeast. I would appreciate it if you could pass on this information to anyone who you think may be interested.

Thank you very much for your help.

Best regards,
Sarah.


Sarah Lambert
Research Scientist/CR1
Institut Curie/CNRS
UMR3348
Bat 110
Centre universitaire Paris-Sud XI
Orsay 91405
France
phone: 00 33 1 69 86 71 91
Fax: 00 33 1 69 86 94 29

http://www.curie.fr/equipe/366
http://www.curie.fr/equipe/366/lang/_gb

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121031/d99cdb64/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Post-doc position.pdf
Type: application/pdf
Size: 251641 bytes
Desc: Post-doc position.pdf
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121031/d99cdb64/attachment-0001.pdf>

From mah79 at cam.ac.uk  Fri Nov  9 14:09:05 2012
From: mah79 at cam.ac.uk (Dr Midori A. Harris)
Date: Fri, 9 Nov 2012 14:09:05 +0000 (GMT)
Subject: [Pombelist] New data and new features on PomBase web site
Message-ID: <Pine.LNX.4.64.1211071527180.32022@hermes-1.csi.cam.ac.uk>

Dear pombe researchers,

We are pleased to announce that we have updated both data and web site 
features for PomBase.

Most importantly, we have added new data types, and upgraded the gene 
pages to display them.

We have also added more annotations of existing data types. The new 
annotations include the first contributions to come in via the new 
community curation system, and we thank the researchers who are 
participating in the initial phase of community curation.

New annotation types:
- Phenotype annotations now use the Fission Yeast Phenotype Ontology 
(FYPO), and include allele details and experimental conditions. With FYPO, 
more detailed phenotypes can be described, and links between terms for 
related phenotypes support improved phenotype searches.

- Many GO annotations now include "annotation extensions" that provide 
additional specificity. For example, extensions may capture the substrate 
of a catalytic activity, the cell cycle phase during which a function or 
process occurs, or any of several other types of supporting information 
for the annotation. For more information about annotation extensions, 
please see the news item on the PomBase web site 
(http://www.pombase.org/news/new-data-and-new-features-pombase-web-site).

You can see these new data types on many gene pages, such as cdc2 
(http://www.pombase.org/spombe/result/SPBC11B10.09) or pka1 
(http://www.pombase.org/spombe/result/SPBC106.10).

Additional new PomBase web site features include colour highlighting for 
retrieved sequences, transcript source data, and version data. A more 
extensive list of web site features is available in the news item 
(http://www.pombase.org/news/new-data-and-new-features-pombase-web-site).

Sincerely yours,
The PomBase Staff




From val at sanger.ac.uk  Mon Nov 12 10:10:59 2012
From: val at sanger.ac.uk (Val Wood)
Date: Mon, 12 Nov 2012 10:10:59 +0000
Subject: [Pombelist] rad/rhp gene names
Message-ID: <50A0CB33.7030904@sanger.ac.uk>


Please note that the primary gene names and synonyms of the following 
rad/rhp genes have been switched to reflect
universal nomenclature as suggested by Matthew O'Connell at pombe 2011, 
and approved by the gene naming committee.

The affected genes, and their current names are:

rad51		synonym rhp51
rad54		synonym rhp54
rad55		synonym rhp55
rad57		synonym rhp57

I thought I had e-mailed pombelist a notification and to see if there 
were any objections, but it appears that I never did this.
Apologies for this omission.

I'll will  update the  products so that they contain the old and the new 
name to reduce any confusion.
e.g. RecA family recombinase Rad51 (previously Rhp51).

Genes will always remain searchable using either name.

Val
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/b60978a4/attachment.htm>

From edoardo.petrini at babraham.ac.uk  Mon Nov 12 10:45:47 2012
From: edoardo.petrini at babraham.ac.uk (edoardo petrini)
Date: Mon, 12 Nov 2012 10:45:47 +0000
Subject: [Pombelist] Covaris Chromatin Sonication
Message-ID: <5501E8B6510D3A4285D425BC20ADF4BCCF4CA9D6@MAIL1.babraham.ac.uk>

Hello everyone,
I have to perform some ChIP experiments and I am now trying to optimise the sonication step. In our Institute we have a Covaris Sonicator that could be very helpful for my purposes, however doesn't seems to work well with chromatin. Does anyone used this machine for chromatin sonication? Does anyone have a protocol to achieve a good sonication (fragments between 500bp and 1kb) with pombe chromatin.
Thank you very much for your help

Kind Regards

Edoardo
The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT Registered Charity No. 1053902.
The information transmitted in this email is directed only to the addressee. If you received this in error, please contact the sender and delete this email from your system. The contents of this e-mail are the views of the sender and do not necessarily represent the views of the Babraham Institute. Full conditions at: www.babraham.ac.uk<http://www.babraham.ac.uk/email_disclaimer.html>


From marti5_kupiec at yahoo.com  Mon Nov 12 10:28:28 2012
From: marti5_kupiec at yahoo.com (Martin Kupiec)
Date: Mon, 12 Nov 2012 02:28:28 -0800 (PST)
Subject: [Pombelist] rad/rhp gene names
In-Reply-To: <50A0CB33.7030904@sanger.ac.uk>
References: <50A0CB33.7030904@sanger.ac.uk>
Message-ID: <1352716108.86230.YahooMailNeo@web162001.mail.bf1.yahoo.com>

What about Rad52 instead of Rad22? Rad52 is called rad52 in all organisms except for Pombe (where Rad52 foci are called rad22 foci); this is a great opportunity to change that too...:-)
Cheers!
Martin


?
Prof. Martin Kupiec
Pasha Gol Chair for Applied Microbiology,
Director, The Constantiner Institute for Molecular Genetics,
Dept. of Molecular Microbiology & Biotechnology,
Tel Aviv University,
Ramat Aviv 69978, Israel.
Phone: 972-3-640-9031
FAX: 972-3-640-9407
E-mail: martin at post.tau.ac.il
http://www.tau.ac.il/lifesci/departments/biotech/members/kupiec/kupiec.html


>________________________________
> From: Val Wood <val at sanger.ac.uk>
>To: pombelist <pombelist at sanger.ac.uk> 
>Sent: Monday, November 12, 2012 12:10 PM
>Subject: [Pombelist] rad/rhp gene names
> 
>
>
>Please note that the primary gene names and synonyms of the
    following rad/rhp genes have been switched to reflect 
>universal nomenclature as suggested by Matthew O'Connell at pombe
    2011, and approved by the gene naming committee.
>
>The affected genes, and their current names are:
>
>rad51		synonym rhp51
rad54		synonym rhp54
rad55		synonym rhp55
rad57		synonym rhp57
I thought I had e-mailed pombelist a notification and to see if there were any objections, but it appears that I never did this.
>Apologies for this omission. 
>
>I'll will? update the? products so that they contain the old and the
    new name to reduce any confusion.
>e.g. RecA family recombinase Rad51 (previously Rhp51).
>
>Genes will always remain searchable using either name. 
>
>Val
>
>_______________________________________________
>Pombelist mailing list
>Pombelist at sanger.ac.uk
>http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/fd55e376/attachment.htm>

From francis.stewart at biotec.tu-dresden.de  Mon Nov 12 10:52:50 2012
From: francis.stewart at biotec.tu-dresden.de (Francis Stewart)
Date: Mon, 12 Nov 2012 11:52:50 +0100
Subject: [Pombelist] rad/rhp gene names
In-Reply-To: <1352716108.86230.YahooMailNeo@web162001.mail.bf1.yahoo.com>
References: <50A0CB33.7030904@sanger.ac.uk>
	<1352716108.86230.YahooMailNeo@web162001.mail.bf1.yahoo.com>
Message-ID: <03170C81-DAFE-47F7-A8A2-74247E5EE594@biotec.tu-dresden.de>

and how about rad6 instead of rhp6?

Maybe there is a whole bunch of these cases

regards

Francis
On 12 Nov 2012, at 11:28, Martin Kupiec wrote:

> What about Rad52 instead of Rad22? Rad52 is called rad52 in all organisms except for Pombe (where Rad52 foci are called rad22 foci); this is a great opportunity to change that too...:-)
> Cheers!
> Martin
> 
>  
> Prof. Martin Kupiec
> Pasha Gol Chair for Applied Microbiology,
> Director, The Constantiner Institute for Molecular Genetics,
> Dept. of Molecular Microbiology & Biotechnology,
> Tel Aviv University,
> Ramat Aviv 69978, Israel.
> Phone: 972-3-640-9031
> FAX: 972-3-640-9407
> E-mail: martin at post.tau.ac.il
> http://www.tau.ac.il/lifesci/departments/biotech/members/kupiec/kupiec.html
> From: Val Wood <val at sanger.ac.uk>
> To: pombelist <pombelist at sanger.ac.uk> 
> Sent: Monday, November 12, 2012 12:10 PM
> Subject: [Pombelist] rad/rhp gene names
> 
> 
> Please note that the primary gene names and synonyms of the following rad/rhp genes have been switched to reflect 
> universal nomenclature as suggested by Matthew O'Connell at pombe 2011, and approved by the gene naming committee.
> 
> The affected genes, and their current names are:
> rad51		synonym rhp51
> rad54		synonym rhp54
> rad55		synonym rhp55
> rad57		synonym rhp57
> I thought I had e-mailed pombelist a notification and to see if there were any objections, but it appears that I never did this.
> Apologies for this omission. 
> 
> I'll will  update the  products so that they contain the old and the new name to reduce any confusion.
> e.g. RecA family recombinase Rad51 (previously Rhp51).
> 
> Genes will always remain searchable using either name. 
> 
> Val
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist


Prof. A. Francis Stewart,
Genomics, Technische Universitaet Dresden
BioInnovationZentrum
Tatzberg 47
01307 Dresden
tel +49-351-46340129 or 30
fax+49-351-46340143
http://www.biotec.tu-dresden.de/research-faculty/stewart/group-page/

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/ac7ba485/attachment.htm>

From j.m.murray at sussex.ac.uk  Mon Nov 12 11:13:35 2012
From: j.m.murray at sussex.ac.uk (Johanne Murray)
Date: Mon, 12 Nov 2012 11:13:35 +0000
Subject: [Pombelist] rad/rhp gene names
In-Reply-To: <03170C81-DAFE-47F7-A8A2-74247E5EE594@biotec.tu-dresden.de>
References: <50A0CB33.7030904@sanger.ac.uk>
	<1352716108.86230.YahooMailNeo@web162001.mail.bf1.yahoo.com>
	<03170C81-DAFE-47F7-A8A2-74247E5EE594@biotec.tu-dresden.de>
Message-ID: <ED1D6A4B094B8344A92769347A85681E06F48B1D@EX-SHA-MBX2.ad.susx.ac.uk>

Rad52 would be good but rhp6 is where it gets complicated.

S.pombe rhp6 is the homologue of S. cerevisiae RAD6 but the original S. pombe rad6 is the homologue of S. cerevesiae RAD18 and the name was changed to rhp18 to clarify that.
Similarly S. pombe rad18 was renamed smc6 to avoid confusion. (The S. cerevisiae homologue was originally called rhc18 but strangely that name didn't catch on)

The original mutants are deposited at the NCYC so by simplifying the names further you run the risk of newcomers to the field working on the wrong mutant.

So while it would be much easier if everything was renamed it only really works when there is not already a history.

Jo



Jo Murray
Genome Damage and Stability Centre,
University of Sussex,
Falmer,
Brighton,
BN1 9RQ

01273 877191
fax 01273 678121

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Francis Stewart
Sent: 12 November 2012 10:53
To: Martin Kupiec
Cc: pombelist
Subject: Re: [Pombelist] rad/rhp gene names

and how about rad6 instead of rhp6?

Maybe there is a whole bunch of these cases

regards

Francis
On 12 Nov 2012, at 11:28, Martin Kupiec wrote:


What about Rad52 instead of Rad22? Rad52 is called rad52 in all organisms except for Pombe (where Rad52 foci are called rad22 foci); this is a great opportunity to change that too...:-)
Cheers!
Martin


Prof. Martin Kupiec
Pasha Gol Chair for Applied Microbiology,
Director, The Constantiner Institute for Molecular Genetics,
Dept. of Molecular Microbiology & Biotechnology,
Tel Aviv University,
Ramat Aviv 69978, Israel.
Phone: 972-3-640-9031
FAX: 972-3-640-9407
E-mail: martin at post.tau.ac.il<mailto:martin at post.tau.ac.il>
http://www.tau.ac.il/lifesci/departments/biotech/members/kupiec/kupiec.html
________________________________
From: Val Wood <val at sanger.ac.uk<mailto:val at sanger.ac.uk>>
To: pombelist <pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>>
Sent: Monday, November 12, 2012 12:10 PM
Subject: [Pombelist] rad/rhp gene names


Please note that the primary gene names and synonyms of the following rad/rhp genes have been switched to reflect
universal nomenclature as suggested by Matthew O'Connell at pombe 2011, and approved by the gene naming committee.

The affected genes, and their current names are:

rad51          synonym rhp51

rad54          synonym rhp54

rad55          synonym rhp55

rad57          synonym rhp57
I thought I had e-mailed pombelist a notification and to see if there were any objections, but it appears that I never did this.
Apologies for this omission.

I'll will  update the  products so that they contain the old and the new name to reduce any confusion.
e.g. RecA family recombinase Rad51 (previously Rhp51).

Genes will always remain searchable using either name.

Val

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://publists.sanger.ac.uk/mailman/listinfo/pombelist
_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://publists.sanger.ac.uk/mailman/listinfo/pombelist


Prof. A. Francis Stewart,

Genomics, Technische Universitaet Dresden

BioInnovationZentrum

Tatzberg 47

01307 Dresden

tel +49-351-46340129 or 30

fax+49-351-46340143

http://www.biotec.tu-dresden.de/research-faculty/stewart/group-page/

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/5b173377/attachment-0001.htm>

From val at sanger.ac.uk  Mon Nov 12 13:06:51 2012
From: val at sanger.ac.uk (Val Wood)
Date: Mon, 12 Nov 2012 13:06:51 +0000
Subject: [Pombelist] rad/rhp gene names
In-Reply-To: <ED1D6A4B094B8344A92769347A85681E06F48B1D@EX-SHA-MBX2.ad.susx.ac.uk>
References: <50A0CB33.7030904@sanger.ac.uk>
	<1352716108.86230.YahooMailNeo@web162001.mail.bf1.yahoo.com>
	<03170C81-DAFE-47F7-A8A2-74247E5EE594@biotec.tu-dresden.de>
	<ED1D6A4B094B8344A92769347A85681E06F48B1D@EX-SHA-MBX2.ad.susx.ac.uk>
Message-ID: <50A0F46B.7050301@sanger.ac.uk>

rad22 - rad52 was in the original list. The should  be changed, it is 
updated in our files but  is currently not updated on the website.

Jo is right, the others are more complicated and could create more 
confusion.

Val


On 12/11/2012 11:13, Johanne Murray wrote:
>
> Rad52 would be good but rhp6 is where it gets complicated.
>
> S.pombe rhp6 is the homologue of S. cerevisiae RAD6 but the original 
> S. pombe rad6 is the homologue of S. cerevesiae RAD18 and the name was 
> changed to rhp18 to clarify that.
>
> Similarly S. pombe rad18 was renamed smc6 to avoid confusion. (The S. 
> cerevisiae homologue was originally called rhc18 but strangely that 
> name didn't catch on)
>
> The original mutants are deposited at the NCYC so by simplifying the 
> names further you run the risk of newcomers to the field working on 
> the wrong mutant.
>
> So while it would be much easier if everything was renamed it only 
> really works when there is not already a history.
>
> Jo
>
> Jo Murray
>
> Genome Damage and Stability Centre,
>
> University of Sussex,
>
> Falmer,
>
> Brighton,
>
> BN1 9RQ
>
> 01273 877191
>
> fax 01273 678121
>
> *From:*pombelist-bounces at sanger.ac.uk 
> [mailto:pombelist-bounces at sanger.ac.uk] *On Behalf Of *Francis Stewart
> *Sent:* 12 November 2012 10:53
> *To:* Martin Kupiec
> *Cc:* pombelist
> *Subject:* Re: [Pombelist] rad/rhp gene names
>
> and how about rad6 instead of rhp6?
>
> Maybe there is a whole bunch of these cases
>
> regards
>
> Francis
>
> On 12 Nov 2012, at 11:28, Martin Kupiec wrote:
>
>
>
> What about Rad52 instead of Rad22? Rad52 is called rad52 in all 
> organisms except for Pombe (where Rad52 foci are called rad22 foci); 
> this is a great opportunity to change that too...:-)
> Cheers!
> Martin
>
> Prof. Martin Kupiec
> Pasha Gol Chair for Applied Microbiology,
> Director, The Constantiner Institute for Molecular Genetics,
> Dept. of Molecular Microbiology & Biotechnology,
> Tel Aviv University,
> Ramat Aviv 69978, Israel.
> Phone: 972-3-640-9031
> FAX: 972-3-640-9407
> E-mail: martin at post.tau.ac.il <mailto:martin at post.tau.ac.il>
> http://www.tau.ac.il/lifesci/departments/biotech/members/kupiec/kupiec.html
>
> ------------------------------------------------------------------------
>
> *From:*Val Wood <val at sanger.ac.uk <mailto:val at sanger.ac.uk>>
> *To:* pombelist <pombelist at sanger.ac.uk <mailto:pombelist at sanger.ac.uk>>
> *Sent:* Monday, November 12, 2012 12:10 PM
> *Subject:* [Pombelist] rad/rhp gene names
>
>
> Please note that the primary gene names and synonyms of the following 
> rad/rhp genes have been switched to reflect
> universal nomenclature as suggested by Matthew O'Connell at pombe 
> 2011, and approved by the gene naming committee.
>
> The affected genes, and their current names are:
>
> rad51          synonym rhp51
> rad54          synonym rhp54
> rad55          synonym rhp55
> rad57          synonym rhp57
>
> I thought I had e-mailed pombelist a notification and to see if there 
> were any objections, but it appears that I never did this.
> Apologies for this omission.
>
> I'll will  update the  products so that they contain the old and the 
> new name to reduce any confusion.
> e.g. RecA family recombinase Rad51 (previously Rhp51).
>
> Genes will always remain searchable using either name.
>
> Val
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk <mailto:Pombelist at sanger.ac.uk>
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk <mailto:Pombelist at sanger.ac.uk>
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
>
> Prof. A. Francis Stewart,
>
> Genomics, Technische Universitaet Dresden
>
> BioInnovationZentrum
>
> Tatzberg 47
>
> 01307 Dresden
>
> tel +49-351-46340129 or 30
>
> fax+49-351-46340143
>
> http://www.biotec.tu-dresden.de/research-faculty/stewart/group-page/
>
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/d8b060dd/attachment.htm>

From kme32 at cam.ac.uk  Mon Nov 12 15:05:36 2012
From: kme32 at cam.ac.uk (Kerstin Ewen)
Date: Mon, 12 Nov 2012 15:05:36 +0000
Subject: [Pombelist] etition against cuts on EU research budget
Message-ID: <0363A672-5442-422A-852E-35088B7345C2@cam.ac.uk>

Dear all,

may I call your attention to this online petition against cuts on EU  
research budget:
http://www.no-cuts-on-research.eu/index.php?file=home.htm

Best wishes

Kerstin
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/4760e63d/attachment-0001.htm>

From shamsunder9 at gmail.com  Tue Nov 13 02:33:39 2012
From: shamsunder9 at gmail.com (Sham Sharma)
Date: Mon, 12 Nov 2012 21:33:39 -0500
Subject: [Pombelist] Covaris Chromatin Sonication
In-Reply-To: <5501E8B6510D3A4285D425BC20ADF4BCCF4CA9D6@MAIL1.babraham.ac.uk>
References: <5501E8B6510D3A4285D425BC20ADF4BCCF4CA9D6@MAIL1.babraham.ac.uk>
Message-ID: <CAP55Fi1Jnyp-MuKLeyO8MV7bttt4bjBC_bi8zrXKMqSrc1XhnQ@mail.gmail.com>

Hi Edoardo

I have used Biogenode sonicator for shearing pombe chromatin and it worked
fine for me. I have no experience with Covaris Sonicator. I used 30 sec ON
and 1 minute OFF for 7-8 cycles. With higher density of cells the number of
cycles could be increased.

Hope this helps.

Sham Sunder Sharma
Thomas Jefferson University, Philadelphia, USA.


On Mon, Nov 12, 2012 at 5:45 AM, edoardo petrini <
edoardo.petrini at babraham.ac.uk> wrote:

> Hello everyone,
> I have to perform some ChIP experiments and I am now trying to optimise
> the sonication step. In our Institute we have a Covaris Sonicator that
> could be very helpful for my purposes, however doesn't seems to work well
> with chromatin. Does anyone used this machine for chromatin sonication?
> Does anyone have a protocol to achieve a good sonication (fragments between
> 500bp and 1kb) with pombe chromatin.
> Thank you very much for your help
>
> Kind Regards
>
> Edoardo
> The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT
> Registered Charity No. 1053902.
> The information transmitted in this email is directed only to the
> addressee. If you received this in error, please contact the sender and
> delete this email from your system. The contents of this e-mail are the
> views of the sender and do not necessarily represent the views of the
> Babraham Institute. Full conditions at: www.babraham.ac.uk<
> http://www.babraham.ac.uk/email_disclaimer.html>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121112/6a9ff986/attachment.htm>

From val at sanger.ac.uk  Tue Nov 13 11:58:41 2012
From: val at sanger.ac.uk (Val Wood)
Date: Tue, 13 Nov 2012 11:58:41 +0000
Subject: [Pombelist] Participate in the fission yeast community curation
	project
Message-ID: <50A235F1.1030406@sanger.ac.uk>

Dear pombe researchers,

The PomBase community curation initiative will soon be officially launched.

Comprehensive manual curation of the S. pombe literature is a primary 
goal of the PomBase project, and among the most important activities of 
the curation staff. Increasing numbers of new publications, and a large 
backlog of older papers, however, mean that complete literature curation 
will require input from the research community to supplement the efforts 
of dedicated professional curators.

We have developed an online curation tool that provides an intuitive 
interface to annotate Gene Ontology details, single gene phenotypes, 
protein modifications and interactions. Multi-gene phenotype curation 
(e.g. double mutants) will soon be included as well.

Participating in community curation is the fastest way to make your 
latest papers visible in PomBase, as the curators must now focus their 
efforts on curating older publications. Annotations made in the 
community curation system will also be rapidly disseminated to other 
databases (e.g. GenBank/ENA/DDBJ, UniProt, BioGRID and GO).

We also invite you to evaluate existing annotations and create new 
annotations for partially curated publications (for example, many papers 
have no phenotype curation).

If you would like to participate, please contact us via 
helpdesk at pombase.org, providing the PubMed ID of the publication(s) you 
would like to curate or update. We will send you a link and specific 
instructions.

Finally, many thanks to our beta testers, who have successfully curated 
papers in the new system:
Dominique Helmlinger (SAGA and tra1), Paul Russell (cnt5), Lynda 
Groocock (hrq1, Boddy lab), Juan Jaun Li (mcb1, Nurse lab), James 
Moseley (pil1), Martin Prevorovsk? (cbf11 & 12, Folk lab), Ursula Fleig 
(sos7), Sophie Martin (sec3), Peter Espenshade (nro1, ofd1), Juan Mata, 
Fransisco Navarro (wee1 and other wee, Nurse lab), Jacky Hayles, and 
especially Ryoko Mandeville (Nurse lab), for tackling a block of older 
papers from the Nurse lab.

The PomBase Staff





-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121113/676127ae/attachment.htm>

From vw253 at cam.ac.uk  Fri Nov 16 14:22:24 2012
From: vw253 at cam.ac.uk (Valerie Wood)
Date: Fri, 16 Nov 2012 14:22:24 +0000
Subject: [Pombelist] rad32/mre11
Message-ID: <50A64C20.2050001@cam.ac.uk>

It has been pointed out that many people use Mre11 instead of Rad32. 
Please elt me know if you work on the gene and you would support (or 
not) the switch of mre11 to the primary name.

Val


From f.z.watts at sussex.ac.uk  Fri Nov 16 14:27:04 2012
From: f.z.watts at sussex.ac.uk (Felicity Watts)
Date: Fri, 16 Nov 2012 14:27:04 +0000
Subject: [Pombelist] rad32/mre11
In-Reply-To: <50A64C20.2050001@cam.ac.uk>
References: <50A64C20.2050001@cam.ac.uk>
Message-ID: <663651AA14BBF0458C20A3EE860DB396123CBD44@EX-SHA-MBX2.ad.susx.ac.uk>

Hi Val

It was my lab that first cloned and published on rad32. I'm happy for it to be Mre11.

Felicity


Dr Felicity Z Watts
Genome Damage and Stability Centre
University of Sussex
Falmer
Brighton
BN1 9QG
UK

Tel: (44) 1273 678257


-----Original Message-----
From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Valerie Wood
Sent: 16 November 2012 14:22
To: pombelist
Subject: [Pombelist] rad32/mre11

It has been pointed out that many people use Mre11 instead of Rad32. 
Please elt me know if you work on the gene and you would support (or 
not) the switch of mre11 to the primary name.

Val

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://publists.sanger.ac.uk/mailman/listinfo/pombelist


From haochen.yu at bc.biol.ethz.ch  Mon Nov 19 10:19:25 2012
From: haochen.yu at bc.biol.ethz.ch (Yu  Haochen)
Date: Mon, 19 Nov 2012 10:19:25 +0000
Subject: [Pombelist] vector for tandem double expression
Message-ID: <CCCFC63C.34D5%haochen.yu@bc.biol.ethz.ch>

Dear pombe folks,

I am in need for a vector where I can express two genes in tandem, driven by either inducible nmt promoters or a low-strength constitutive promoters. I am planning to eventually the construct at ura4 locus. Does such a system already exists or perhaps there is a similar one I can modify?

Thanks a lot in advance!

Haochen


--
Dr. Haochen (Jerry) Yu
Institut of Biochemistry
ETH Zurich
HPM D14.1
Schafmattstr. 18
8093 Zurich, Switzerland
Phone: +41 44 632 3015
Email: haochen.yu at bc.biol.ethz.ch<mailto:haochen.yu at bc.biol.ethz.ch>


From hoffmacs at bc.edu  Mon Nov 19 13:00:32 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Mon, 19 Nov 2012 08:00:32 -0500
Subject: [Pombelist] vector for tandem double expression
In-Reply-To: <CCCFC63C.34D5%haochen.yu@bc.biol.ethz.ch>
References: <CCCFC63C.34D5%haochen.yu@bc.biol.ethz.ch>
Message-ID: <FD8F78DB-EBEB-43A9-8181-426D190813C1@bc.edu>

I cannot think of one off hand, however we have recently published a plasmid for a different purpose that may be useful in doing this.  Plasmid pUL57 has three selectable markers on it.  

http://www.ncbi.nlm.nih.gov/pubmed/22198627


It has lys7 driven by its own promoter.
It has ura5 that is divergently transcribed from lys7 using the tif471 promoter.
It has S. cerevisiae LEU2 that is transcribed from the SV40 promoter.

-------------- next part --------------
A non-text attachment was scrubbed...
Name: PastedGraphic-3.tiff
Type: image/tiff
Size: 389064 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121119/1bf6bb6c/attachment-0001.tiff>
-------------- next part --------------


There are unique restriction sites in all three genes that could be used to linearize the plasmid for gap repair transformation to replace an ORF with your gene of interest that has been amplified by PCR to have flanking sequences that target the insert to the plasmid.  We usually only include about 45 nt in the oligo for targeting when we are using a plasmid rather than targeting to the chromosome.  The ura5 gene can also be used with 5FOA to find inserts to the plasmid. We have already done this a couple of times with no problems.

Once you have replaced two ORFs with the ones you want, you can integrate this plasmid to either the ars1 locus or the lys7 locus on chromosome 1.

Let me know if you would like this plasmid along with a strain carrying mutations in ura5, lys7, and leu1.

Cheers,

Charlie Hoffman


On Nov 19, 2012, at 5:19 AM, Yu Haochen wrote:

> Dear pombe folks,
> 
> I am in need for a vector where I can express two genes in tandem, driven by either inducible nmt promoters or a low-strength constitutive promoters. I am planning to eventually the construct at ura4 locus. Does such a system already exists or perhaps there is a similar one I can modify?
> 
> Thanks a lot in advance!
> 
> Haochen
> 
> 
> --
> Dr. Haochen (Jerry) Yu
> Institut of Biochemistry
> ETH Zurich
> HPM D14.1
> Schafmattstr. 18
> 8093 Zurich, Switzerland
> Phone: +41 44 632 3015
> Email: haochen.yu at bc.biol.ethz.ch<mailto:haochen.yu at bc.biol.ethz.ch>
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"




From dddubey2003 at gmail.com  Tue Nov 20 14:27:51 2012
From: dddubey2003 at gmail.com (Dharanidhar Dubey)
Date: Tue, 20 Nov 2012 19:57:51 +0530
Subject: [Pombelist] our paper on stress-induced duplex destabilization
	(SIDD) analysis of S. pombe replication origins.
Message-ID: <000001cdc72b$3c07d5c0$b4178140$@com>

Dear pombe list members,

 

Following is the URL of our paper on stress-induced duplex destabilization
(SIDD) analysis of S. pombe replication origins published in BMC Research
Notes on November 19, 2012:

 

Article URL http://www.biomedcentral.com/1756-0500/5/643

 

I hope, you may find it interesting. In case of questions, kindly contact me
at dddubey2003 at gmail.com.

 

All the best,

 

DD Dubey

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121120/a4e21c92/attachment.htm>

From ramab at stanford.edu  Wed Nov 21 20:37:19 2012
From: ramab at stanford.edu (Rama Balakrishnan)
Date: Wed, 21 Nov 2012 12:37:19 -0800
Subject: [Pombelist] SGD Downtime (Servers Moving to New Data Center)
Message-ID: <55612890-BE38-4EE2-BFBB-21E58B77AD6A@stanford.edu>

Dear Colleague,

This is a message alerting you of planned downtime for the Saccharomyces Genome Database resources for the week of November 27, 2012.

Many SGD tools and resources will be unavailable from Tuesday afternoon, November 27th, until Thursday, November 29th because we will be moving our entire computing operation to a newly opened data center on Stanford campus.  This new data center will provides a very robust environment including a more secure facility, a faster network, and a backup generator in the event of a power failure.  There is also plenty of room for expansion.  All of these features were missing from our current computing facility.

During the downtime you will be able to search the main SGD database at <http://www.yeastgenome.org>.  However, many tools and pages, including the following, will be unavailable: Pathway Tools, YeastMine, Downloads/FTP, SPELL, GBrowse, Textpresso, and the wiki. 

We apologize for the inconvenience and thank you for your patience and understanding as we make this major improvement at SGD to enhance our service to research for the future.

Please contact us at sgd-helpdesk at lists.stanford.edu if you have any questions or concerns.

-Mike Cherry


J. Michael Cherry, Ph.D.         |   Associate Professor (Research)
Department of Genetics           |   Stanford University
Postal: 300 Pasteur Drive        |   Stanford, CA 94305-5120
Office: 1501 S. California Ave.  |   Palo Alto, CA  94304-5577
Voice:  650-723-7541 (office)    |   Email: cherry at stanford.edu
Web: www.yeastgenome.org         |   Web: www.geneontology.org

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121121/42352f8a/attachment.htm>

From vw253 at cam.ac.uk  Fri Nov 23 16:19:08 2012
From: vw253 at cam.ac.uk (Valerie Wood)
Date: Fri, 23 Nov 2012 16:19:08 +0000
Subject: [Pombelist] rad32/mre11
Message-ID: <50AFA1FC.5010906@cam.ac.uk>


So far, 9 researchers who have published on rad32/mre11 have approved 
this change, including Felicity who first published  on rad32. Nobody 
has objected, therefore rad32 now has the primary name mre11, and the 
synonym rad32. I have made the change in the database and it should be 
visible in a week or two.

Best wishes,

Val





From n.bibi at ucl.ac.uk  Thu Nov 29 16:52:24 2012
From: n.bibi at ucl.ac.uk (Bibi, Nazia)
Date: Thu, 29 Nov 2012 16:52:24 +0000
Subject: [Pombelist] Protocol for transmembrane protein extraction and
	westernblotting
Message-ID: <A512DF788050D34F8BAB53D4CB7AA00004C06768@DB3PRD0104MB108.eurprd01.prod.exchangelabs.com>

Dear Pombe community,

I am trying to extract and do the western blotting for proteins that are predicted to be transmembrane. So far, I could not get any success and seems that protein extraction is failing. It would be much appreciated if anyone can send me the optimised protocol for transmembrane protein extraction and western blotting.

Cheers,

Nazia


-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121129/c3452224/attachment.htm>

From f.marsellach-castellvi at ucl.ac.uk  Fri Nov 30 18:40:02 2012
From: f.marsellach-castellvi at ucl.ac.uk (Marsellach-Castellvi, Xavier)
Date: Fri, 30 Nov 2012 18:40:02 +0000
Subject: [Pombelist] Mitochondrial transfromation in S. pombe
Message-ID: <F534A583-4661-439D-93A0-2EAF20D0E900@ucl.ac.uk>

Dear Pombe Researchers,
Does anybody have experience in Biolistic transformation in S. pombe to insert markers into the S. pombe mitochondria? This technique has been successfully applied in S. cerevisiae, but to my knowledge this techniques has not been yet established in S. pombe.
I would appreciate if anyone that has experience in this technique could share his/her expertise.
Thanks,
Francesc Xavier Marsellach-Castellvi
Department of Genetics, Evolution and Environment
The Darwin Building
University College London
London WC1E 6BT, UK
--------------------------------------------------
Phone: +44(0)203-108-16-03
Fax: +44 (0)208-679-70-96
Web: http://www.bahlerlab.info/




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121130/d6a9c0ac/attachment.htm>

From bonnefoy at cgm.cnrs-gif.fr  Sat Dec  1 14:11:37 2012
From: bonnefoy at cgm.cnrs-gif.fr (bonnefoy at cgm.cnrs-gif.fr)
Date: Sat, 01 Dec 2012 15:11:37 +0100
Subject: [Pombelist] Mitochondrial transfromation in S. pombe
In-Reply-To: <F534A583-4661-439D-93A0-2EAF20D0E900@ucl.ac.uk>
References: <F534A583-4661-439D-93A0-2EAF20D0E900@ucl.ac.uk>
Message-ID: <20121201151137.hqvltueeyoc0sc4c@mail.cgm.cnrs-gif.fr>

Hi Xavier,

I have use biolistic transformation of mitochondria extensively in S.  
cerevisiae and in my lab we are (still) trying to set up the technique  
for S. pombe. We managed to send DNA into S. pombe mitochondria and we  
observed recombination events, however so far we couldn't create a  
stable modified mitochondrial DNA (the recombination of the resident  
mitochondrial genome with the exogenous marker was never 100% in our  
hands). But we keep trying!

If you would like to discuss this matter further please fell free to  
write back.

Best wishes, Nathalie



Quoting "Marsellach-Castellvi, Xavier" <f.marsellach-castellvi at ucl.ac.uk>:

> Dear Pombe Researchers,
> Does anybody have experience in Biolistic transformation in S. pombe  
>  to insert markers into the S. pombe mitochondria? This technique  
> has  been successfully applied in S. cerevisiae, but to my knowledge  
> this  techniques has not been yet established in S. pombe.
> I would appreciate if anyone that has experience in this technique   
> could share his/her expertise.
> Thanks,
> Francesc Xavier Marsellach-Castellvi
> Department of Genetics, Evolution and Environment
> The Darwin Building
> University College London
> London WC1E 6BT, UK
> --------------------------------------------------
> Phone: +44(0)203-108-16-03
> Fax: +44 (0)208-679-70-96
> Web: http://www.bahlerlab.info/
>
>
>
>
>





From forsburg at usc.edu  Wed Dec  5 22:56:25 2012
From: forsburg at usc.edu (SLForsburg)
Date: Wed, 5 Dec 2012 14:56:25 -0800
Subject: [Pombelist] Looking for red Cnp1
Message-ID: <82479ECE-B4D8-4C86-B747-E850BD40C049@usc.edu>

Hi folks,
We are doing some live cell studies and need a red Cnp1.  Does anyone have one?

Thanks!


susan


 
{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 






From val at sanger.ac.uk  Fri Dec  7 11:02:25 2012
From: val at sanger.ac.uk (Val Wood)
Date: Fri, 07 Dec 2012 11:02:25 +0000
Subject: [Pombelist] Important notice if you have used GO enrichment tools
	since September.
Message-ID: <50C1CCC1.1030403@sanger.ac.uk>


In the period since September and yesterday our GO dataset was reduced 
because our annotations with IEA (inferred from electronic annotation)  
evidence codes  had expired as they were over one year old,  and our new 
pipeline was not in place.

This could affect enrichment exercises performed in this period as ~169 
genes are annotated with a process only via the IEA evidence code ( 
mainly where the IEA annotation captures what is currently known). Other 
gene products also have some IEA annotation which might improve the 
results of an enrichment exercise.

If you have used  a GO enrichment tool i.e GO term finder 
http://go.princeton.edu/cgi-bin/GOTermFinder in the period
you may  wish to repeat your analysis with the new improved dataset.

Best wishes,

Val

(The current dataset  has 34,947 annotations using manual evidence 
codes, and 4775 IEA annotations)


From pjh at sanger.ac.uk  Mon Dec 10 07:23:18 2012
From: pjh at sanger.ac.uk (Paul Hunt)
Date: Mon, 10 Dec 2012 07:23:18 -0000
Subject: [Pombelist] Schizosaccharomyces pombe
Message-ID: <800005B5BBF29A46AC6B4E0F6DD1B0D5017F6224@vmexchsrv.internal.sanger.ac.uk>

Hi

   We have received the request below in the Archives section , if
anybody is able to help it will be much appreciated.

 

 

I'm currently performing research on the karyopherin Kap123 from a
variety of different fungal homologs and was wondering how I would go
about obtaining the cDNA for Kap123 from Schizosaccharomyces pombe. The
GI number for the gene is 6801316.



Thanks

 

Paul Hunt

Sample Management

The Wellcome Trust Sanger Institute

Genome Campus

Hinxton

Cambridge

CB10 1SA

 

TEL: 01223 <TEL:01223>  492324

FAX:01223 495353

 

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121210/9b4fba45/attachment.htm>

From hoffmacs at bc.edu  Mon Dec 10 13:16:17 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Mon, 10 Dec 2012 08:16:17 -0500
Subject: [Pombelist] Schizosaccharomyces pombe
In-Reply-To: <800005B5BBF29A46AC6B4E0F6DD1B0D5017F6224@vmexchsrv.internal.sanger.ac.uk>
References: <800005B5BBF29A46AC6B4E0F6DD1B0D5017F6224@vmexchsrv.internal.sanger.ac.uk>
Message-ID: <5B678D0B-90A4-40E8-8F18-09FDF9AB8359@bc.edu>

When I need a cDNA, I just get it by PCR from one of the two size-selected cDNA libraries that we have in the lab.  

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"




On Dec 10, 2012, at 2:23 AM, Paul Hunt wrote:

> Hi
>    We have received the request below in the Archives section , if anybody is able to help it will be much appreciated.
>  
>  
> I'm currently performing research on the karyopherin Kap123 from a variety of different fungal homologs and was wondering how I would go about obtaining the cDNA for Kap123 from Schizosaccharomyces pombe. The GI number for the gene is 6801316.
> 
> Thanks
>  
> Paul Hunt
> Sample Management
> The Wellcome Trust Sanger Institute
> Genome Campus
> Hinxton
> Cambridge
> CB10 1SA
>  
> TEL: 01223 492324
> FAX:01223 495353
>  
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist



-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121210/6659926e/attachment.htm>

From Dominique.Helmlinger at crbm.cnrs.fr  Thu Dec 13 13:57:32 2012
From: Dominique.Helmlinger at crbm.cnrs.fr (Dominique Helmlinger)
Date: Thu, 13 Dec 2012 14:57:32 +0100
Subject: [Pombelist] Group Leader positions in Montpellier, France
Message-ID: <50C9DECC.4040803@crbm.cnrs.fr>

Dear Pombe Community,

Please find attached an open call for group leader positions we have 
launched at the Macromolecular Biochemistry Research Center (CRBM), in 
Montpellier, France. We would be grateful if you could bring the 
announcementto the attention of potential candidatesand post it in your 
department or institute.

Cheers,

Dom Helmlinger

-- 

Dom Helmlinger, PhD

CRBM - CNRS UMR5237
1919 Route de Mende
Cedex 5
34293 Montpellier
France

tel: +33/0-434-359-548
email: dhelmlinger at crbm.cnrs.fr
web: https://sites.google.com/site/thehelmlingerlab/

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121213/cab39223/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: CRBM-call.pdf
Type: application/octet-stream
Size: 2027217 bytes
Desc: not available
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121213/cab39223/attachment-0001.obj>

From JXW180 at bham.ac.uk  Fri Dec 14 12:33:39 2012
From: JXW180 at bham.ac.uk (Jianming Wang)
Date: Fri, 14 Dec 2012 12:33:39 +0000
Subject: [Pombelist] Problems of plating cells on YEA media
Message-ID: <9C0C995DCC5E7B429EFAC473E846407C77AD166D@mbx05.adf.bham.ac.uk>

Dear Pombe Community,

I have been trying to do the cell growth rate experiment by plating samples taken from YES culture at different time points on YEA plates. However, the growth rate results I got from counting the number of colonies grown on YEA plates does not match the growth pattern reflected by the OD. It seems that I have problems of plating cells on the plates. I would appreciate if someone has suggestions on how to improve this kind of experiments or some detailed protocols on doing similar experiments like cell survival of acute exposure to hydroxyurea? Thanks!


Jianming


Jianming Wang

School Of Biosciences

University of Birmingham

Edgbaston

Birmingham, B15 2TT

UK


From hoffmacs at bc.edu  Fri Dec 14 13:01:11 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Fri, 14 Dec 2012 08:01:11 -0500
Subject: [Pombelist] Problems of plating cells on YEA media
In-Reply-To: <9C0C995DCC5E7B429EFAC473E846407C77AD166D@mbx05.adf.bham.ac.uk>
References: <9C0C995DCC5E7B429EFAC473E846407C77AD166D@mbx05.adf.bham.ac.uk>
Message-ID: <26A13878-A6DF-4A76-88A4-C53682F5B3CB@bc.edu>

Have you looked at the cels or counted using a hemocytometer?  Your OD could include dead cells or your colony count could be low due to cell clumping.  

I would trust a hemocytometer count as the most direct assessment of growth rate that has the fewest questionable assumptions.  

Charlie Hoffman


On Dec 14, 2012, at 7:33 AM, Jianming Wang wrote:

> Dear Pombe Community,
> 
> I have been trying to do the cell growth rate experiment by plating samples taken from YES culture at different time points on YEA plates. However, the growth rate results I got from counting the number of colonies grown on YEA plates does not match the growth pattern reflected by the OD. It seems that I have problems of plating cells on the plates. I would appreciate if someone has suggestions on how to improve this kind of experiments or some detailed protocols on doing similar experiments like cell survival of acute exposure to hydroxyurea? Thanks!
> 
> 
> Jianming
> 
> 
> Jianming Wang
> 
> School Of Biosciences
> 
> University of Birmingham
> 
> Edgbaston
> 
> Birmingham, B15 2TT
> 
> UK
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"





From forsburg at usc.edu  Fri Dec 14 16:49:19 2012
From: forsburg at usc.edu (SLForsburg)
Date: Fri, 14 Dec 2012 08:49:19 -0800
Subject: [Pombelist] Problems of plating cells on YEA media
In-Reply-To: <26A13878-A6DF-4A76-88A4-C53682F5B3CB@bc.edu>
References: <9C0C995DCC5E7B429EFAC473E846407C77AD166D@mbx05.adf.bham.ac.uk>
	<26A13878-A6DF-4A76-88A4-C53682F5B3CB@bc.edu>
Message-ID: <C4349E54-9179-4514-B48A-F2E0E6A8DBC2@usc.edu>

I agree with Charlie.  OD is a measure of cell mass;  cells that elongate will have a higher OD than the same number of cells that are small, and it can't tell live from dead.   It is only an approximation of cell number.  When in doubt, a hemocytometer is the way to go.




On Dec 14, 2012, at 5:01 AM, Charlie Hoffman wrote:

> Have you looked at the cels or counted using a hemocytometer?  Your OD could include dead cells or your colony count could be low due to cell clumping.  
> 
> I would trust a hemocytometer count as the most direct assessment of growth rate that has the fewest questionable assumptions.  
> 
> Charlie Hoffman
> 
> 
> On Dec 14, 2012, at 7:33 AM, Jianming Wang wrote:
> 
>> Dear Pombe Community,
>> 
>> I have been trying to do the cell growth rate experiment by plating samples taken from YES culture at different time points on YEA plates. However, the growth rate results I got from counting the number of colonies grown on YEA plates does not match the growth pattern reflected by the OD. It seems that I have problems of plating cells on the plates. I would appreciate if someone has suggestions on how to improve this kind of experiments or some detailed protocols on doing similar experiments like cell survival of acute exposure to hydroxyurea? Thanks!
>> 
>> 
>> Jianming
>> 
>> 
>> Jianming Wang
>> 
>> School Of Biosciences
>> 
>> University of Birmingham
>> 
>> Edgbaston
>> 
>> Birmingham, B15 2TT
>> 
>> UK
>> 
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
> 
> Charlie Hoffman
> hoffmacs at bc.edu
> "I'd rather be fission"
> 
> 
> 
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist

 
{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 






From JXW180 at bham.ac.uk  Sun Dec 16 22:40:04 2012
From: JXW180 at bham.ac.uk (Jianming Wang)
Date: Sun, 16 Dec 2012 22:40:04 +0000
Subject: [Pombelist] Problems of plating cells on YEA media
In-Reply-To: <C4349E54-9179-4514-B48A-F2E0E6A8DBC2@usc.edu>
References: <9C0C995DCC5E7B429EFAC473E846407C77AD166D@mbx05.adf.bham.ac.uk>
	<26A13878-A6DF-4A76-88A4-C53682F5B3CB@bc.edu>,
	<C4349E54-9179-4514-B48A-F2E0E6A8DBC2@usc.edu>
Message-ID: <9C0C995DCC5E7B429EFAC473E846407C77AD7BA3@mbx06.adf.bham.ac.uk>

Dear Professor Forsburg and Professor Hoffman,

Thanks a lot for your suggestions. The spot assay suggested by Professor Hoffman is a smart way to assess the mutants sensitivity to drug treatment. While I also want to check the cell viability treated by HU for different time period. For that aim, I hope to do exactly the same experiment as Professor Forsburg did in the Figure 1G in your recently published nice paper in Mol Cell Biol (Sabatinos SA et al, 2012). So could I have the detailed protocol that you did this experiment? Besides, I am worried that if I did not do it well for the NO HU control experiment in the similar way, my viability assay would be less convincing. Thanks in advance.

Best wishes,

Jianming Wang   


________________________________________
From: SLForsburg [forsburg at usc.edu]
Sent: 14 December 2012 16:49
To: Charlie Hoffman
Cc: Jianming Wang; pombelist at sanger.ac.uk
Subject: Re: [Pombelist] Problems of plating cells on YEA media

I agree with Charlie.  OD is a measure of cell mass;  cells that elongate will have a higher OD than the same number of cells that are small, and it can't tell live from dead.   It is only an approximation of cell number.  When in doubt, a hemocytometer is the way to go.




On Dec 14, 2012, at 5:01 AM, Charlie Hoffman wrote:

> Have you looked at the cels or counted using a hemocytometer?  Your OD could include dead cells or your colony count could be low due to cell clumping.
>
> I would trust a hemocytometer count as the most direct assessment of growth rate that has the fewest questionable assumptions.
>
> Charlie Hoffman
>
>
> On Dec 14, 2012, at 7:33 AM, Jianming Wang wrote:
>
>> Dear Pombe Community,
>>
>> I have been trying to do the cell growth rate experiment by plating samples taken from YES culture at different time points on YEA plates. However, the growth rate results I got from counting the number of colonies grown on YEA plates does not match the growth pattern reflected by the OD. It seems that I have problems of plating cells on the plates. I would appreciate if someone has suggestions on how to improve this kind of experiments or some detailed protocols on doing similar experiments like cell survival of acute exposure to hydroxyurea? Thanks!
>>
>>
>> Jianming
>>
>>
>> Jianming Wang
>>
>> School Of Biosciences
>>
>> University of Birmingham
>>
>> Edgbaston
>>
>> Birmingham, B15 2TT
>>
>> UK
>>
>> _______________________________________________
>> Pombelist mailing list
>> Pombelist at sanger.ac.uk
>> http://publists.sanger.ac.uk/mailman/listinfo/pombelist
>
> Charlie Hoffman
> hoffmacs at bc.edu
> "I'd rather be fission"
>
>
>
>
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist


{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu
www.pombe.net

From pluskal at oist.jp  Tue Dec 18 02:18:43 2012
From: pluskal at oist.jp (Tomas Pluskal)
Date: Tue, 18 Dec 2012 02:18:43 +0000
Subject: [Pombelist] Measuring cell concentration in liquid cultures
Message-ID: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>

Dear pombe researchers,

I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore.

We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.

I would appreciate to hear about your experience using other machines, particularly if anyone knows a good cell counter especially suitable for pombe cultures in terms of reliability and high throughput.

Best regards,

Tomas


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
WWW: https://groups.oist.jp/g0
TEL: +81-98-966-8684
Fax: +81-98-966-2890

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/e1a021fb/attachment.htm>

From Conrad.Nieduszynski at nottingham.ac.uk  Tue Dec 18 09:49:49 2012
From: Conrad.Nieduszynski at nottingham.ac.uk (Conrad Nieduszynski)
Date: Tue, 18 Dec 2012 09:49:49 +0000
Subject: [Pombelist] British Yeast Group meeting - registration now open
Message-ID: <887EC5EB-DDDC-4A5D-A07A-F6C21BDA5931@exmail.nottingham.ac.uk>

An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/3334fe46/attachment.htm>

From hoffmacs at bc.edu  Tue Dec 18 13:32:57 2012
From: hoffmacs at bc.edu (Charlie Hoffman)
Date: Tue, 18 Dec 2012 08:32:57 -0500
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
References: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
Message-ID: <86B349B5-D43A-4072-A882-7F135B7BDADD@bc.edu>

We are not high tech in my lab for this.  We simply use a hemacytometer.  The advantage to this is that it makes you actually look at the cells, which provides more information than just cell numbers.  

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"


On Dec 17, 2012, at 9:18 PM, Tomas Pluskal wrote:

> Dear pombe researchers,
> 
> I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore. 
> 
> We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.
> 
> I would appreciate to hear about your experience using other machines, particularly if anyone knows a good cell counter especially suitable for pombe cultures in terms of reliability and high throughput. 
> 
> Best regards,
> 
> Tomas
> 
> 
> ===============================================
> Tomas Pluskal
> G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
> 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
> WWW: https://groups.oist.jp/g0
> TEL: +81-98-966-8684
> Fax: +81-98-966-2890
> 
> _______________________________________________
> Pombelist mailing list
> Pombelist at sanger.ac.uk
> http://publists.sanger.ac.uk/mailman/listinfo/pombelist




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/f70e74a6/attachment.htm>

From onigen at bio.ku.dk  Tue Dec 18 13:41:50 2012
From: onigen at bio.ku.dk (Olaf Nielsen)
Date: Tue, 18 Dec 2012 14:41:50 +0100
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
References: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
Message-ID: <8F3D8FD74B8BAE4488865C1AD2F45C9C1381B6@mailserver11.akizci.ku.dk>

Dear Tomas,

 

We use the NC-3000 Nucleocounter, which is highly recommendable, see:

 

http://www.chemometec.com/en-GB/Products/NucleoCounter-NC-3000.aspx

 

 

Best regards,

 

Olaf

 

 

----

Olaf Nielsen, Professor, Vice-Head of Dept.                                    

Cell Cycle and Genome Stability Group                     

Department of Biology, University of Copenhagen      

Ole Maal?es Vej 5, DK-2200 Copenhagen K, Denmark

e-mail: onigen at bio.ku.dk / Tel: +45 3532 2102                                                                                      

 

 

 

 

From: pombelist-bounces at sanger.ac.uk [mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Tomas Pluskal
Sent: 18. december 2012 03:19
To: pombelist at sanger.ac.uk
Subject: [Pombelist] Measuring cell concentration in liquid cultures

 

Dear pombe researchers, 

 

I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore. 

 

We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.

 

I would appreciate to hear about your experience using other machines, particularly if anyone knows a good cell counter especially suitable for pombe cultures in terms of reliability and high throughput. 

 

Best regards,

 

Tomas

 

 

===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan

WWW: https://groups.oist.jp/g0
TEL: +81-98-966-8684
Fax: +81-98-966-2890

 

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/d48acc17/attachment.htm>

From c.rallis at ucl.ac.uk  Tue Dec 18 13:44:39 2012
From: c.rallis at ucl.ac.uk (Rallis, Babis)
Date: Tue, 18 Dec 2012 13:44:39 +0000
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
References: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
Message-ID: <9A4E5886046F2340B527EB98EB0132E90CD3BE31@DB3PRD0104MB167.eurprd01.prod.exchangelabs.com>

Hi Tomas
We use a Z1 coulter particle counter from Beckman Coulter.
20 ul of culture diluted in 10 or 20 ml of isotone buffer is a usual practice.
The time required per sample is around 10 s but it requires flushing the system
so probably timing could be same as in your case. No clogging.
Best
Babis

--------------------------------------------------
Charalampos (Babis) Rallis
Department of Genetics, Evolution and Environment
and UCL Cancer Institute
University College London
Darwin Building, Gower Street,
WC1E 6BT, London,UK
--------
 P: +44 (0)20 3108 1606
 F: +44 (0)20 7679 7096
 www.bahlerlab.info
--------------------------------------------------
________________________________
From: pombelist-bounces at sanger.ac.uk [pombelist-bounces at sanger.ac.uk] on behalf of Tomas Pluskal [pluskal at oist.jp]
Sent: 18 December 2012 02:18
To: pombelist at sanger.ac.uk
Subject: [Pombelist] Measuring cell concentration in liquid cultures

Dear pombe researchers,

I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore.

We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.

I would appreciate to hear about your experience using other machines, particularly if anyone knows a good cell counter especially suitable for pombe cultures in terms of reliability and high throughput.

Best regards,

Tomas


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
WWW: https://groups.oist.jp/g0
TEL: +81-98-966-8684
Fax: +81-98-966-2890

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/33bd44c4/attachment-0001.htm>

From cagataytarhan at yahoo.com  Tue Dec 18 14:03:54 2012
From: cagataytarhan at yahoo.com (cagatay tarhan)
Date: Tue, 18 Dec 2012 06:03:54 -0800 (PST)
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <86B349B5-D43A-4072-A882-7F135B7BDADD@bc.edu>
References: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
	<86B349B5-D43A-4072-A882-7F135B7BDADD@bc.edu>
Message-ID: <1355839434.4673.YahooMailNeo@web161801.mail.bf1.yahoo.com>

Hi from Turkey, We use Cedex HiREs Analyser and here is the link for it:

http://www.roche-applied-science.com/sis/innovatis/index.jsp?id=innovatis_020300



________________________________
 From: Charlie Hoffman <hoffmacs at bc.edu>
To: Tomas Pluskal <pluskal at oist.jp> 
Cc: "pombelist at sanger.ac.uk" <pombelist at sanger.ac.uk> 
Sent: Tuesday, December 18, 2012 3:32 PM
Subject: Re: [Pombelist] Measuring cell concentration in liquid cultures
 

We are not high tech in my lab for this. ?We simply use a hemacytometer. ?The advantage to this is that it makes you actually look at the cells, which provides more information than just cell numbers. ?

Charlie Hoffman
hoffmacs at bc.edu
"I'd rather be fission"


On Dec 17, 2012, at 9:18 PM, Tomas Pluskal wrote:

Dear pombe researchers, 
>
>
>I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore.?
>
>
>We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s?for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.
>
>
>I would appreciate to hear about your experience using other machines, particularly if anyone knows a good?cell counter especially suitable for pombe cultures in terms of reliability and high throughput.?
>
>
>Best regards,
>
>
>Tomas
>
>
>
>
>===============================================
>Tomas Pluskal
>G0 Cell Unit, Okinawa Institute of Science?and Technology Graduate University
>1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
>WWW:?https://groups.oist.jp/g0
>TEL: +81-98-966-8684
>Fax: +81-98-966-2890
>
_______________________________________________
>Pombelist mailing list
>Pombelist at sanger.ac.uk
>http://publists.sanger.ac.uk/mailman/listinfo/pombelist




_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://publists.sanger.ac.uk/mailman/listinfo/pombelist
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/f1d74c91/attachment.htm>

From echang1 at bcm.edu  Tue Dec 18 17:34:09 2012
From: echang1 at bcm.edu (Chang, Eric C)
Date: Tue, 18 Dec 2012 11:34:09 -0600
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <9A4E5886046F2340B527EB98EB0132E90CD3BE31@DB3PRD0104MB167.eurprd01.prod.exchangelabs.com>
Message-ID: <CCF5FE3C.24FCC%echang1@bcm.tmc.edu>

For most "growth" experiments, the critical parameter that we are measuring is the growth rate in order to calculate the doubling time.  Thus, even though your method does not directly measure dividing cells, as long as the instrument, or your technique, is consistent with a high degree of reproducibility, you will get the same growth rate, regardless of what instrument/method you use.  Each method has its inherited errors but these will be evened out over the course of a growth curve experiment.  Thus the cheapest and fastest way of doing this is to use an instrument that measure the presence of "particles" in solution at a wave length of 600 nm, which is essential white visible light.  A good old spectrophotometer can be used for this, obviously.  We have found this portable single wavelength device for $700 USD (called Ultraspec 10, see attached) that does nothing but measure cell density.  It is easy and simply to use and can be moved around from bench to bench.  With all optical based measurement, the instrument shows the best linearity between OD of 0.1-0.6 or so so it is important to dilute the cell to be within this range to get an accurate measurement.

Measuring cell density and colony number by plating are two slightly different experiments, although if one were to focus only on the "growth rate," the two methods should still yield the same results.  In particular, the colony formation assay directly measure the number of cells that can form colonies, while the optical density method measures the balance between growth and death/dormancy.  To properly plate the cell, one thing that we find it important is to make sure that the cells are evenly dispersed, as much as possible by vortexing vigorously, and EGTA/EDTA can be added to help, provided that they do not affect the growth of your cells.  We also typically plate the cells in quadruplicate to better control variation.


--
Best,

Eric Chang
N1130 (inside N1130)/x83519

From: <Rallis>, Babis <c.rallis at ucl.ac.uk<mailto:c.rallis at ucl.ac.uk>>
Date: Tuesday, December 18, 2012 7:44 AM
To: Tomas Pluskal <pluskal at oist.jp<mailto:pluskal at oist.jp>>, "pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>" <pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>>
Subject: Re: [Pombelist] Measuring cell concentration in liquid cultures

Hi Tomas
We use a Z1 coulter particle counter from Beckman Coulter.
20 ul of culture diluted in 10 or 20 ml of isotone buffer is a usual practice.
The time required per sample is around 10 s but it requires flushing the system
so probably timing could be same as in your case. No clogging.
Best
Babis

--------------------------------------------------
Charalampos (Babis) Rallis
Department of Genetics, Evolution and Environment
and UCL Cancer Institute
University College London
Darwin Building, Gower Street,
WC1E 6BT, London,UK
--------
 P: +44 (0)20 3108 1606
 F: +44 (0)20 7679 7096
 www.bahlerlab.info
--------------------------------------------------
________________________________
From: pombelist-bounces at sanger.ac.uk<mailto:pombelist-bounces at sanger.ac.uk> [pombelist-bounces at sanger.ac.uk<mailto:pombelist-bounces at sanger.ac.uk>] on behalf of Tomas Pluskal [pluskal at oist.jp<mailto:pluskal at oist.jp>]
Sent: 18 December 2012 02:18
To: pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>
Subject: [Pombelist] Measuring cell concentration in liquid cultures

Dear pombe researchers,

I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore.

We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.

I would appreciate to hear about your experience using other machines, particularly if anyone knows a good cell counter especially suitable for pombe cultures in terms of reliability and high throughput.

Best regards,

Tomas


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
WWW: https://groups.oist.jp/g0
TEL: +81-98-966-8684
Fax: +81-98-966-2890

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/6a60b75d/attachment-0001.htm>
-------------- next part --------------
A non-text attachment was scrubbed...
Name: Ultraspec 10.jpeg
Type: image/jpeg
Size: 244663 bytes
Desc: Ultraspec 10.jpeg
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/6a60b75d/attachment-0001.jpeg>

From forsburg at usc.edu  Tue Dec 18 19:02:59 2012
From: forsburg at usc.edu (SLForsburg)
Date: Tue, 18 Dec 2012 11:02:59 -0800
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <CCF5FE3C.24FCC%echang1@bcm.tmc.edu>
References: <CCF5FE3C.24FCC%echang1@bcm.tmc.edu>
Message-ID: <794A2E26-7A83-42EE-BE4E-B37A8B7E4ED2@usc.edu>

I'm going to disagree  with this:
>  
> Measuring cell density and colony number by plating are two slightly different experiments, although if one were to focus only on the "growth rate," the two methods should still yield the same results.   

Actually, they are quite different experiments.

We have plenty of mutants that will continue to "grow" (increase OD) for a few hours, but are essentially dying and unable to form colonies. If we are treating with a drug like HU that induces elongation, or a cdc mutant, the increase in OD may be reflecting increasing cell size and NOT increasing cell number.  Or, cells may be dividing (increasing OD and cell number) but not be capable of forming colonies. 

 If we do a viability assay, we always count them with a hemocytometer, never via OD.   

I think of it as the Southern California Freeway model of execution points:  your car may continue  to coast some distance after you run out of gas, but you aren't really moving very far.
 
--susan



{} {} {} {} {} {} {} {} {} {} {} {} {} {}
S L Forsburg  PhD, Professor
Molecular & Computational Biology 
University of Southern California
Ray R Irani Hall
1050 Childs Way, RRI 201B
Los Angeles, CA 90089-2910

vox:  213-740-7342
fax:   213-740-8631
forsburg at usc.edu 
www.pombe.net 




-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121218/63bda02e/attachment.htm>

From pluskal at oist.jp  Wed Dec 19 01:03:07 2012
From: pluskal at oist.jp (Tomas Pluskal)
Date: Wed, 19 Dec 2012 01:03:07 +0000
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <1355839434.4673.YahooMailNeo@web161801.mail.bf1.yahoo.com>
References: <1879A92C-8BC5-46D8-B261-0207D9D2CBE6@oist.jp>
	<86B349B5-D43A-4072-A882-7F135B7BDADD@bc.edu>,
	<1355839434.4673.YahooMailNeo@web161801.mail.bf1.yahoo.com>
Message-ID: <5EA8F66B-42F1-458C-95E6-74C01B8009AC@oist.jp>

Thank you for all the answers! We will check the instruments you suggested.

Just wondering - can any of these instruments measure multiple samples automatically, e.g. from a 96-well plate?  I find a lot of time wasted by manually sonicating and switching the samples for measurement.

Tomas


On 18. 12. 2012, at 23:03, "cagatay tarhan" <cagataytarhan at yahoo.com<mailto:cagataytarhan at yahoo.com>> wrote:

Hi from Turkey, We use Cedex HiREs Analyser and here is the link for it:

http://www.roche-applied-science.com/sis/innovatis/index.jsp?id=innovatis_020300

________________________________
From: Charlie Hoffman <hoffmacs at bc.edu<mailto:hoffmacs at bc.edu>>
To: Tomas Pluskal <pluskal at oist.jp<mailto:pluskal at oist.jp>>
Cc: "pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>" <pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>>
Sent: Tuesday, December 18, 2012 3:32 PM
Subject: Re: [Pombelist] Measuring cell concentration in liquid cultures

We are not high tech in my lab for this.  We simply use a hemacytometer.  The advantage to this is that it makes you actually look at the cells, which provides more information than just cell numbers.

Charlie Hoffman
hoffmacs at bc.edu<mailto:hoffmacs at bc.edu>
"I'd rather be fission"


On Dec 17, 2012, at 9:18 PM, Tomas Pluskal wrote:

Dear pombe researchers,

I am wondering what equipment do you use to measure the cell concentration of liquid cultures. In our lab we use the Sysmex CDA-500, a rather old machine that I believe is not produced anymore.

We usually dilute 10 or 100 ul of cell culture into 10 ml of saline buffer, sonicate for 10 s and measure on the Sysmex. The measurement takes about 15 s, therefore the complete procedure takes about 30 s for one sample, if everything goes well. But the machine often gets clogged and requires cleaning.

I would appreciate to hear about your experience using other machines, particularly if anyone knows a good cell counter especially suitable for pombe cultures in terms of reliability and high throughput.

Best regards,

Tomas


===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan
WWW: https://groups.oist.jp/g0
TEL: +81-98-966-8684
Fax: +81-98-966-2890

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://publists.sanger.ac.uk/mailman/listinfo/pombelist





_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk<mailto:Pombelist at sanger.ac.uk>
http://publists.sanger.ac.uk/mailman/listinfo/pombelist

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121219/ede6d761/attachment.htm>

From Erik.Boye at rr-research.no  Wed Dec 19 10:56:37 2012
From: Erik.Boye at rr-research.no (Erik Boye)
Date: Wed, 19 Dec 2012 11:56:37 +0100
Subject: [Pombelist] Measuring cell concentration in liquid cultures
In-Reply-To: <CCF5FE3C.24FCC%echang1@bcm.tmc.edu>
References: <9A4E5886046F2340B527EB98EB0132E90CD3BE31@DB3PRD0104MB167.eurprd01.prod.exchangelabs.com>
	<CCF5FE3C.24FCC%echang1@bcm.tmc.edu>
Message-ID: <AFE5B2464F1EB547BBACE62CCE8E402001D1157A@mr3k6001.ad.medicalresearch.no>

Dear Thomas, 

What you choose to measure is dependent upon your particular experiment
and exactly what it is that you wish to measure. In a balanced,
steady-state culture I would not care to measure the number of particles
(cells) per ml or the number of colony-forming units, but rather measure
the rise in optical density. Up to an OD of about 0.3 (600 nm light) the
rise in OD should be strictly exponential, as are the other parameters.
At higher ODs the cells start sense nutrient changes and change
morphology and metabolism, so the different parameters behave
differently. 

If you wish to monitor your parameter under changing conditions, the
picture is different. Then you need to measure exactly the parameter
that you are looking for, whether it is cell size or viability or number
of particles or a combination of different parameters. You can be sure
that one parameter does not necessarily follow another. A simple example
would be to shift a cdcts to the restrictive temperature, in which case
you might see nothing on the OD curve, but cell size and number of
particles and number of viable cells will dramatically increase/decline.


 

Your original question was to measure the number of cells per ml. To do
that you absolutely need a "particle counter" and you have got some
suggestions. Make sure that you can detect your particles/cells
separately from background noise in your system. The Coulter counters -
or a similar orifice-based principle - usually work very well. You
should also be aware that if you have a flow cytometer available, they
might do the trick. Just check that the pump is stable enough for your
measurements, i.e. do repeated experiments in the beginning.

 

Best regards,

Erik

 

From: pombelist-bounces at sanger.ac.uk
[mailto:pombelist-bounces at sanger.ac.uk] On Behalf Of Chang, Eric C
Sent: 18. desember 2012 18:34
To: Rallis, Babis; Tomas Pluskal; pombelist at sanger.ac.uk
Subject: Re: [Pombelist] Measuring cell concentration in liquid cultures

 

For most "growth" experiments, the critical parameter that we are
measuring is the growth rate in order to calculate the doubling time.
Thus, even though your method does not directly measure dividing cells,
as long as the instrument, or your technique, is consistent with a high
degree of reproducibility, you will get the same growth rate, regardless
of what instrument/method you use.  Each method has its inherited errors
but these will be evened out over the course of a growth curve
experiment.  Thus the cheapest and fastest way of doing this is to use
an instrument that measure the presence of "particles" in solution at a
wave length of 600 nm, which is essential white visible light.  A good
old spectrophotometer can be used for this, obviously.  We have found
this portable single wavelength device for $700 USD (called Ultraspec
10, see attached) that does nothing but measure cell density.  It is
easy and simply to use and can be moved around from bench to bench.
With all optical based measurement, the instrument shows the best
linearity between OD of 0.1-0.6 or so so it is important to dilute the
cell to be within this range to get an accurate measurement.

 

Measuring cell density and colony number by plating are two slightly
different experiments, although if one were to focus only on the "growth
rate," the two methods should still yield the same results.  In
particular, the colony formation assay directly measure the number of
cells that can form colonies, while the optical density method measures
the balance between growth and death/dormancy.  To properly plate the
cell, one thing that we find it important is to make sure that the cells
are evenly dispersed, as much as possible by vortexing vigorously, and
EGTA/EDTA can be added to help, provided that they do not affect the
growth of your cells.  We also typically plate the cells in
quadruplicate to better control variation.

 

 

--

Best,

 

Eric Chang

N1130 (inside N1130)/x83519

 

From: <Rallis>, Babis <c.rallis at ucl.ac.uk>
Date: Tuesday, December 18, 2012 7:44 AM
To: Tomas Pluskal <pluskal at oist.jp>, "pombelist at sanger.ac.uk"
<pombelist at sanger.ac.uk>
Subject: Re: [Pombelist] Measuring cell concentration in liquid cultures

 

Hi Tomas
We use a Z1 coulter particle counter from Beckman Coulter.
20 ul of culture diluted in 10 or 20 ml of isotone buffer is a usual
practice.
The time required per sample is around 10 s but it requires flushing the
system 
so probably timing could be same as in your case. No clogging.
Best
Babis

 

--------------------------------------------------

Charalampos (Babis) Rallis

Department of Genetics, Evolution and Environment 

and UCL Cancer Institute 

University College London 

Darwin Building, Gower Street, 

WC1E 6BT, London,UK

--------

 P: +44 (0)20 3108 1606

 F: +44 (0)20 7679 7096

 www.bahlerlab.info

--------------------------------------------------

________________________________

From: pombelist-bounces at sanger.ac.uk [pombelist-bounces at sanger.ac.uk] on
behalf of Tomas Pluskal [pluskal at oist.jp]
Sent: 18 December 2012 02:18
To: pombelist at sanger.ac.uk
Subject: [Pombelist] Measuring cell concentration in liquid cultures

Dear pombe researchers, 

 

I am wondering what equipment do you use to measure the cell
concentration of liquid cultures. In our lab we use the Sysmex CDA-500,
a rather old machine that I believe is not produced anymore. 

 

We usually dilute 10 or 100 ul of cell culture into 10 ml of saline
buffer, sonicate for 10 s and measure on the Sysmex. The measurement
takes about 15 s, therefore the complete procedure takes about 30 s for
one sample, if everything goes well. But the machine often gets clogged
and requires cleaning.

 

I would appreciate to hear about your experience using other machines,
particularly if anyone knows a good cell counter especially suitable for
pombe cultures in terms of reliability and high throughput. 

 

Best regards,

 

Tomas

 

 

===============================================
Tomas Pluskal
G0 Cell Unit, Okinawa Institute of Science and Technology Graduate
University
1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan

WWW: https://groups.oist.jp/g0
TEL: +81-98-966-8684
Fax: +81-98-966-2890

 

-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://publists.sanger.ac.uk/pipermail/pombelist/attachments/20121219/35396d7e/attachment-0001.htm>

From bogus@does.not.exist.com  Mon Sep 10 10:27:41 2012
From: bogus@does.not.exist.com ()
Date: Mon, 10 Sep 2012 09:27:41 -0000
Subject: No subject
Message-ID: <mailman.3.1359470433.2428.pombelist@sanger.ac.uk>

be a reference out there that confirms it.

best

Mikel

Mikel Zaratiegui
Assistant Professor
Department of Molecular Biology and Biochemistry
Rutgers, the State University of New Jersey
Nelson Research Building, A139
604 Allison Rd
Piscataway NJ 08854
(732) 445-1497

On Jan 29, 2013, at 7:00 AM, <pombelist-request at sanger.ac.uk<mailto:pombeli=
st-request at sanger.ac.uk>>
 wrote:

Send Pombelist mailing list submissions to
pombelist at sanger.ac.uk<mailto:pombelist at sanger.ac.uk>

To subscribe or unsubscribe via the World Wide Web, visit
http://publists.sanger.ac.uk/mailman/listinfo/pombelist
or, via email, send a message with subject or body 'help' to
pombelist-request at sanger.ac.uk

You can reach the person managing the list at
pombelist-owner at sanger.ac.uk

When replying, please edit your Subject line so it is more specific
than "Re: Contents of Pombelist digest..."


Today's Topics:

  1. leu1.32 and ade6-M216 sequence (Amar)
  2. Re: leu1.32 and ade6-M216 sequence (Fleck,Oliver)


----------------------------------------------------------------------

Message: 1
Date: Mon, 28 Jan 2013 15:45:08 -0500
From: Amar <klara at mail.nih.gov>
Subject: [Pombelist] leu1.32 and ade6-M216 sequence
To: <pombelist at sanger.ac.uk>
Message-ID: <p06020421cd2c92a03da4@[129.43.18.12]>
Content-Type: text/plain; charset=3D"us-ascii"; format=3Dflowed

Dear Pombe community,

Please someone send the sequences or source of pombe leu1, leu1.32,
and ade6 and ade6-M216 alleles.

Thanks much,

Amar Klar
--
Amar Klar Ph.D
Section Head
Gene Regulation and Chromosome Biology laboratory
National Cancer Institute at Frederick
7th Street, Ft. Detrick
P.O. Box B, Frederick, MD 21702
Phone 301 846 5916
Fax 301 846 6911
E-mail: klara at mail.nih.gov



------------------------------

Message: 2
Date: Tue, 29 Jan 2013 09:30:26 +0000
From: "Fleck,Oliver" <o.fleck at bangor.ac.uk>
Subject: Re: [Pombelist] leu1.32 and ade6-M216 sequence
To: Amar <klara at mail.nih.gov>
Cc: pombelist at sanger.ac.uk
Message-ID: <510796B2.7050605 at bangor.ac.uk>
Content-Type: text/plain; charset=3DISO-8859-1; format=3Dflowed

Dear Amar,

The ade6-M216 mutation can be found in Szankasi et al. 1988 J. Mol.
Biol. 204: 917-925.

http://www.ncbi.nlm.nih.gov/pubmed/3221399

Best wishes,

Oliver


Amar wrote:
Dear Pombe community,

Please someone send the sequences or source of pombe leu1, leu1.32, and
ade6 and ade6-M216 alleles.

Thanks much,

Amar Klar


--
Oliver Fleck

North West Cancer Research Fund Institute
School of Biological Sciences
Bangor University
Brambell Building
Bangor LL57 2UW
UK

Phone: +44 (0)1248 38 8189
--
Rhif Elusen Gofrestredig / Registered Charity No. 1141565

Gall y neges e-bost hon, ac unrhyw atodiadau a anfonwyd gyda hi,
gynnwys deunydd cyfrinachol ac wedi eu bwriadu i'w defnyddio'n unig
gan y sawl y cawsant eu cyfeirio ato (atynt). Os ydych wedi derbyn y
neges e-bost hon trwy gamgymeriad, rhowch wybod i'r anfonwr ar
unwaith a dil?wch y neges. Os na fwriadwyd anfon y neges atoch chi,
rhaid i chi beidio ? defnyddio, cadw neu ddatgelu unrhyw wybodaeth a
gynhwysir ynddi. Mae unrhyw farn neu safbwynt yn eiddo i'r sawl a'i
hanfonodd yn unig  ac nid yw o anghenraid yn cynrychioli barn
Prifysgol Bangor. Nid yw Prifysgol Bangor yn gwarantu
bod y neges e-bost hon neu unrhyw atodiadau yn rhydd rhag firysau neu
100% yn ddiogel. Oni bai fod hyn wedi ei ddatgan yn uniongyrchol yn
nhestun yr e-bost, nid bwriad y neges e-bost hon yw ffurfio contract
rhwymol - mae rhestr o lofnodwyr awdurdodedig ar gael o Swyddfa
Cyllid Prifysgol Bangor.  www.bangor.ac.uk

This email and any attachments may contain confidential material and
is solely for the use of the intended recipient(s).  If you have
received this email in error, please notify the sender immediately
and delete this email.  If you are not the intended recipient(s), you
must not use, retain or disclose any information contained in this
email.  Any views or opinions are solely those of the sender and do
not necessarily represent those of Bangor University.
Bangor University does not guarantee that this email or
any attachments are free from viruses or 100% secure.  Unless
expressly stated in the body of the text of the email, this email is
not intended to form a binding contract - a list of authorised
signatories is available from the Bangor University Finance
Office.  www.bangor.ac.uk



------------------------------

_______________________________________________
Pombelist mailing list
Pombelist at sanger.ac.uk
http://publists.sanger.ac.uk/mailman/listinfo/pombelist

End of Pombelist Digest, Vol 9, Issue 15
****************************************



From bogus@does.not.exist.com  Mon Sep 10 10:27:41 2012
From: bogus@does.not.exist.com ()
Date: Mon, 10 Sep 2012 09:27:41 -0000
Subject: No subject
Message-ID: <mailman.4.1359471741.2428.pombelist@sanger.ac.uk>

be a reference out there that confirms it.<br>
<br>
best<br>
<br>
Mikel<br>
<br>
Mikel Zaratiegui<br>
Assistant Professor<br>
Department of Molecular Biology and Biochemistry<br>
Rutgers, the State University of New Jersey<br>
Nelson Research Building, A139<br>
604 Allison Rd<br>
Piscataway NJ 08854<br>
<a href=3D"tel:%28732%29%20445-1497" value=3D"+17324451497" target=3D"_blan=
k">(732) 445-1497</a><br>
<br>
On Jan 29, 2013, at 7:00 AM, &lt;<a href=3D"mailto:pombelist-request at sanger=
.ac.uk" target=3D"_blank">pombelist-request at sanger.ac.uk</a>&lt;mailto:<a h=
ref=3D"mailto:pombelist-request at sanger.ac.uk" target=3D"_blank">pombelist-r=
equest at sanger.ac.uk</a>&gt;&gt;<br>


=A0wrote:<br>
<br>
Send Pombelist mailing list submissions to<br>
<a href=3D"mailto:pombelist at sanger.ac.uk" target=3D"_blank">pombelist at sange=
r.ac.uk</a>&lt;mailto:<a href=3D"mailto:pombelist at sanger.ac.uk" target=3D"_=
blank">pombelist at sanger.ac.uk</a>&gt;<br>
<br>
To subscribe or unsubscribe via the World Wide Web, visit<br>
<a href=3D"http://publists.sanger.ac.uk/mailman/listinfo/pombelist" target=
=3D"_blank">http://publists.sanger.ac.uk/mailman/listinfo/pombelist</a><br>
or, via email, send a message with subject or body &#39;help&#39; to<br>
<a href=3D"mailto:pombelist-request at sanger.ac.uk" target=3D"_blank">pombeli=
st-request at sanger.ac.uk</a><br>
<br>
You can reach the person managing the list at<br>
<a href=3D"mailto:pombelist-owner at sanger.ac.uk" target=3D"_blank">pombelist=
-owner at sanger.ac.uk</a><br>
<br>
When replying, please edit your Subject line so it is more specific<br>
than &quot;Re: Contents of Pombelist digest...&quot;<br>
<br>
<br>
Today&#39;s Topics:<br>
<br>
=A0 1. leu1.32 and ade6-M216 sequence (Amar)<br>
=A0 2. Re: leu1.32 and ade6-M216 sequence (Fleck,Oliver)<br>
<br>
<br>
----------------------------------------------------------------------<br>
<br>
Message: 1<br>
Date: Mon, 28 Jan 2013 15:45:08 -0500<br>
From: Amar &lt;<a href=3D"mailto:klara at mail.nih.gov" target=3D"_blank">klar=
a at mail.nih.gov</a>&gt;<br>
Subject: [Pombelist] leu1.32 and ade6-M216 sequence<br>
To: &lt;<a href=3D"mailto:pombelist at sanger.ac.uk" target=3D"_blank">pombeli=
st at sanger.ac.uk</a>&gt;<br>
Message-ID: &lt;p06020421cd2c92a03da4@[129.43.18.12]&gt;<br>
Content-Type: text/plain; charset=3D&quot;us-ascii&quot;; format=3Dflowed<b=
r>
<br>
Dear Pombe community,<br>
<br>
Please someone send the sequences or source of pombe leu1, leu1.32,<br>
and ade6 and ade6-M216 alleles.<br>
<br>
Thanks much,<br>
<br>
Amar Klar<br>
--<br>
Amar Klar Ph.D<br>
Section Head<br>
Gene Regulation and Chromosome Biology laboratory<br>
National Cancer Institute at Frederick<br>
7th Street, Ft. Detrick<br>
P.O. Box B, Frederick, MD 21702<br>
Phone <a href=3D"tel:301%20846%205916" value=3D"+13018465916" target=3D"_bl=
ank">301 846 5916</a><br>
Fax <a href=3D"tel:301%20846%206911" value=3D"+13018466911" target=3D"_blan=
k">301 846 6911</a><br>
E-mail: <a href=3D"mailto:klara at mail.nih.gov" target=3D"_blank">klara at mail.=
nih.gov</a><br>
<br>
<br>
<br>
------------------------------<br>
<br>
Message: 2<br>
Date: Tue, 29 Jan 2013 09:30:26 +0000<br>
From: &quot;Fleck,Oliver&quot; &lt;<a href=3D"mailto:o.fleck at bangor.ac.uk" =
target=3D"_blank">o.fleck at bangor.ac.uk</a>&gt;<br>
Subject: Re: [Pombelist] leu1.32 and ade6-M216 sequence<br>
To: Amar &lt;<a href=3D"mailto:klara at mail.nih.gov" target=3D"_blank">klara@=
mail.nih.gov</a>&gt;<br>
Cc: <a href=3D"mailto:pombelist at sanger.ac.uk" target=3D"_blank">pombelist at s=
anger.ac.uk</a><br>
Message-ID: &lt;<a href=3D"mailto:510796B2.7050605 at bangor.ac.uk" target=3D"=
_blank">510796B2.7050605 at bangor.ac.uk</a>&gt;<br>
Content-Type: text/plain; charset=3DISO-8859-1; format=3Dflowed<br>
<br>
Dear Amar,<br>
<br>
The ade6-M216 mutation can be found in Szankasi et al. 1988 J. Mol.<br>
Biol. 204: 917-925.<br>
<br>
<a href=3D"http://www.ncbi.nlm.nih.gov/pubmed/3221399" target=3D"_blank">ht=
tp://www.ncbi.nlm.nih.gov/pubmed/3221399</a><br>
<br>
Best wishes,<br>
<br>
Oliver<br>
<br>
<br>
Amar wrote:<br>
Dear Pombe community,<br>
<br>
Please someone send the sequences or source of pombe leu1, leu1.32, and<br>
ade6 and ade6-M216 alleles.<br>
<br>
Thanks much,<br>
<br>
Amar Klar<br>
<br>
<br>
--<br>
Oliver Fleck<br>
<br>
North West Cancer Research Fund Institute<br>
School of Biological Sciences<br>
Bangor University<br>
Brambell Building<br>
Bangor LL57 2UW<br>
UK<br>
<br>
Phone: <a href=3D"tel:%2B44%20%280%291248%2038%208189" value=3D"+4412483881=
89" target=3D"_blank">+44 (0)1248 38 8189</a><br>
--<br>
Rhif Elusen Gofrestredig / Registered Charity No. 1141565<br>
<br>
Gall y neges e-bost hon, ac unrhyw atodiadau a anfonwyd gyda hi,<br>
gynnwys deunydd cyfrinachol ac wedi eu bwriadu i&#39;w defnyddio&#39;n unig=
<br>
gan y sawl y cawsant eu cyfeirio ato (atynt). Os ydych wedi derbyn y<br>
neges e-bost hon trwy gamgymeriad, rhowch wybod i&#39;r anfonwr ar<br>
unwaith a dil?wch y neges. Os na fwriadwyd anfon y neges atoch chi,<br>
rhaid i chi beidio ? defnyddio, cadw neu ddatgelu unrhyw wybodaeth a<br>
gynhwysir ynddi. Mae unrhyw farn neu safbwynt yn eiddo i&#39;r sawl a&#39;i=
<br>
hanfonodd yn unig =A0ac nid yw o anghenraid yn cynrychioli barn<br>
Prifysgol Bangor. Nid yw Prifysgol Bangor yn gwarantu<br>
bod y neges e-bost hon neu unrhyw atodiadau yn rhydd rhag firysau neu<br>
100% yn ddiogel. Oni bai fod hyn wedi ei ddatgan yn uniongyrchol yn<br>
nhestun yr e-bost, nid bwriad y neges e-bost hon yw ffurfio contract<br>
rhwymol - mae rhestr o lofnodwyr awdurdodedig ar gael o Swyddfa<br>
Cyllid Prifysgol Bangor. =A0<a href=3D"http://www.bangor.ac.uk" target=3D"_=
blank">www.bangor.ac.uk</a><br>
<br>
This email and any attachments may contain confidential material and<br>
is solely for the use of the intended recipient(s). =A0If you have<br>
received this email in error, please notify the sender immediately<br>
and delete this email. =A0If you are not the intended recipient(s), you<br>
must not use, retain or disclose any information contained in this<br>
email. =A0Any views or opinions are solely those of the sender and do<br>
not necessarily represent those of Bangor University.<br>
Bangor University does not guarantee that this email or<br>
any attachments are free from viruses or 100% secure. =A0Unless<br>
expressly stated in the body of the text of the email, this email is<br>
not intended to form a binding contract - a list of authorised<br>
signatories is available from the Bangor University Finance<br>
Office. =A0<a href=3D"http://www.bangor.ac.uk" target=3D"_blank">www.bangor=
.ac.uk</a><br>
<br>
<br>
<br>
------------------------------<br>
<br>
_______________________________________________<br>
Pombelist mailing list<br>
<a href=3D"mailto:Pombelist at sanger.ac.uk" target=3D"_blank">Pombelist at sange=
r.ac.uk</a><br>
<a href=3D"http://publists.sanger.ac.uk/mailman/listinfo/pombelist" target=
=3D"_blank">http://publists.sanger.ac.uk/mailman/listinfo/pombelist</a><br>
<br>
End of Pombelist Digest, Vol 9, Issue 15<br>
****************************************<br>
<br>
<br>
_______________________________________________<br>
Pombelist mailing list<br>
<a href=3D"mailto:Pombelist at sanger.ac.uk" target=3D"_blank">Pombelist at sange=
r.ac.uk</a><br>
<a href=3D"http://publists.sanger.ac.uk/mailman/listinfo/pombelist" target=
=3D"_blank">http://publists.sanger.ac.uk/mailman/listinfo/pombelist</a><br>
<br>
<br>
</blockquote></div><br><br clear=3D"all"><br>-- <br>Antonia Lock, PhD<br>
PomBase Biocurator, <a href=3D"http://www.pombase.org" target=3D"_blank">ht=
tp://www.pombase.org</a><br>
Department of Genetics, Evolution and Environment,<br>
The Darwin Building,<br>
University College London<br>
London WC1E 6BT, UK

--14dae9341165daafd204d46eacdc--