I am currently trying to make a ts allele of a gene that I am studying
in S. cerevisiae. I am doing this using in the following way:
I have a haploid null mutant (for my gene) kept alive by a functional
copy of my gene on a URA3 ADE3 2 micron plasmid.
I did PCR mutagenesis of my gene and cotransformed the PCR fragments
with a linearized TRP1 CEN plasmid that has DNA homologous to the ends
of my PCR fragments, into the strain above....the PCR frag gets
homologously recombined with this plasmid to reconstitute the vector.
I plate transformants at 37C and pick colonies that stay red (i.e. need
to keep the first plasmid) to sterile microtiter dishes. 1100 red
colonies were then "frogged" to YEPG. From YEPG, these where then
frogged to SDG plus 5FOA (1 g/ L) with 50 ug/ml uracil and other
appropriate amino acids at 25C and 37C.
I have obtained a number of colonies that grow in a
temperature-sensitive fashion on the 5FOA plates (2-3 rounds of
restreaking on 5FOA to make sure the first plasmid is gone). However,
so far, NONE of these are also ts when streaked on YEPG. My quesstion
is, has anyone run this kind of screen and seen this before? If yes, at
what rate did you these false positives? Finally, do you have an
explanation as to what could be going on that causes these cells to be
ts on 5FOA, but not rich media? I feel that I have successfully cured
the first plasmid as the cells are now white....I am currently checking
these strains for ura auxotrophy to make sure.
Thanks for taking the time to read this, any hints/tips or advice is
taron at uiuc.edu