In article <2gv183$3ic at hub.ucsb.edu>, Pa Gan <upagan at mcl.ucsb.edu> wrote:
> I'm currently working on developing a TS mutant library within a mutant
> strain of S. cerevisae, however, using my current methods, i am not
> generating the ammounts of TS mutants for my library.
> My current method is to perform mutagenesis on my strain with EMS, and
> grow them at 30C, then replica plate them and grow them at 37C and look
> for colonies which haven't grown. This method seems pretty straight
> forward however i have gotten very few TS mutants from my strain. Does anyone know of a better method for developing a TS mutant library? Thanks much
> in advance for your replies! Pat.
>gan at lifesci.ucsb.edu>upagan at mcl.mcl.ucsb.edu>First, you should probably grow the mutagenized yeast at a lower
temperature, something like 25C, before replica-plating to 37C. It is
often the case that mutants that don't grow at 37C are also not too
happy at 30C.
Second, check the killing frequency by plating dilutions of your yeast
culture before and after mutagenesis. I usually aim for about 50%
killing. This results in about 1/2500 red colonies, which gives you
an idea of the frequency of knocking out either ADE1 or ADE2.
stearns at leland.stanford.edu