Help me! / Yeast transformation in Two-Hybrid screening

[Joe Boutell]

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

Hello!
We've done several screens using CLONTECHS kit, and have picked up a 
couple of tips that combined together increased our transformation 
efficiencies 10-fold (although not as high as the 200 thousand 
cfu/microgram you're looking for).
1) after inoculation with the overnight culture, grow the 300ml culture 
up to an OD600 of 0.6 - this may take longer than the 3 hours 
recommended.
2) instead of doimg the transformation in one big tube, split it up into 
10-20 1.5ml eppendorfs (i know it takes longer, but it works). Don't just 
make up a big transformation and split it, but make up each small scale 
transformation separately.
Be wary that we do this using the original MATCHMAKER system, using 
different yeast strains to the one you are using.
Another place to look for yeast transformation help is the homepage of 
Gietz's lab. Sorry, but i've lost the address - try a net search.
Cheers,
Joe Boutell.