Methods for developing TS mutant libraries in S. cerevisae
dan at cubmol.bio.columbia.edu
Tue Jan 11 18:07:20 EST 1994
In article <2gv183$3ic at hub.ucsb.edu>, Pa Gan <upagan at mcl.ucsb.edu> wrote:
> 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
Try growing up at room temperature, then replica'ing to 37. The
difference between 30 and 37 might be too slight. Also check the accuracy
of your incubators. Sometimes thermometers are not accurate, and sometimes
incubators have a temperature gradient in them. Another possibility is that
the temperature varies in your incubators. We had an air shaker that would
go from 30 to 37+ every morning from 6 to 8. The sun was hitting it only
during those hours.
I was making a collection of ts strains once, and I got about 0.1% to
1% in the first screen.
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