Help me! / Yeast transformation in Two-Hybrid screening

Michael Hamilton mhamilto at gpu.srv.ualberta.ca
Thu Aug 22 00:27:21 EST 1996


In article <naganot-1908961822530001 at molnb03.bri.niigata-u.ac.jp>,
naganot at bri.niigata-u.ac.jp (Takashi Nagano) wrote:

> I am now doing Two-Hybrid screening with CLONTECH's MATCHMAKER 2 system
> (yeast: CG1945, bait plasmid: pAS2-1) and pGAD10-based library. My
> problem is too low transformation efficiency in sequential
> transformation. I have tried on several protocols including one in
> CLONTECH's manual, but the efficiency is no more than 4000cfu/microgram
> plasmid so far. I hear that 200 thousand cfu/microgram plasmid can be
> achieved by some researchers. Someone who knows an optimal or better
> protocol and/or SD medium recipe for transforming CG1945 yeast, please
> teach me how they are!
> Thank you very much for your help. 
> 
> My e-mail address is : naganot at bri.niigata-u.ac.jp
> 
> Takashi Nagano

The best protocol that I know of is the modified LiAcetate protocol put
together by the Geitz lab at the University of Manitoba.  I don't have the
location handy, but do a Web search using saccharomyces, geitz and
manitoba as keywords and you ought to get to it.   
   One note:  I don't have much success with their listed method for
preparing the carrier DNA (you'll know what I mean when you get there) I
get better results using an older method ( in the Scheistl and Geitz
reference)

   I also haven't gotten the results that Geitz claims, but I haven't
spent as much time at it as they have either.  

   Luck



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