[Pombelist] library complementation

[Antony Carr]

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
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