[Pombelist] Diploid pombe + high efficiency HR by 2-step PCR ...

[Dr Michael-Christopher Keogh]

Burcin,

Re. the transformation issue we generally transform with ~400bp overlapping homology as created by a 2-step PCR protocol. Lots of colonies and >90% clone of interest ...

All current lab protocols available at:  http://sites.google.com/site/mckeogh2/protocols

Specific of interest is attached ..

Michael  ...




On Dec 16, 2010, at 4:59 AM, burcin altun wrote:

> 
> Dear all,
> I am a master student at molecular biology and genetics departmant at istanbul universty/Turkey and now i am doing my master thesis at ICGEB/Italy. I have some questions.
> * I am working with diploid pombe (972 h- x MeiB102) and i want to check them again to be sure that i have diploid. I used phloxin B but unhealhty haploid cells can also take phloxinB so it isnt a certain way to check them. I want to perform PCR but i dont know which location in genome can be target? How can i perform pcr to check this mutant?
> * Does anyone send me colony PCR protocol because i tried different methots but i have problem about efficiency of PCR.
> * And for knock-out i used 90bp but i didnt take positive results. So do you think what can i do? Do i have to increase homology region  in my cassette?
> Looking forward to hear from you soon 
> Thanking you in advance
>  
> Burcin ALTUN (MSc. Student)
> *Istanbul University
>  Department of Molecular Biology and Genetics
>  Vezneciler, 34118 Istanbul TURKEY
> *ICGEB
> International Centre for Genetic Engineering and Biotechnology (ICGEB)
> Padriciano 99, 34012 Trieste, Italy
> E-mail:   altun at icgeb.org
> Phone:  +39-340-8613728
>  
> 
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____________________

Dr Michael-Christopher Keogh
Assistant Professor
Department of Cell Biology (Chanin 415)
Albert Einstein College of Medicine
1300 Morris Park Avenue
Bronx, NY 10461
USA

Tel:  (718) 430-8796 / 2944 (Office / Lab)
Fax: (718) 430-8574
Web: http://mckeogh.googlepages.com

Email: michael.keogh at einstein.yu.edu



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