Perhaps someone monitoring this group can explain the following
observations:
I have received a S. cerevisiae strain (Mat a rad52::LEU2 trp1, ade2-1
his7, ura3-52 ise1 top1-1 leu2) in which I have transformed two plasmids:
One is a 2 micron plasmid carrying the TRP1 marker. The second plasmid is
a CEN plasmid carrying the URA3 marker. I am trying to grow this stain in
liquid culture under conditions that will induce expression from the GAL1
gene promoter that is upstream of a gene on the URA3 "marked" CEN plasmid.
I am using a published protocol for growing this particular strain--In the
published protocol the strain is tranformed with a URA3 "marked" 2 micron
plasmid--under galactose inducing conidtions (It seemed to be a
straight forward, standard protocol to me).
The strain is grown to midlog phase at 30 degrees C with shaking in
minimal glucose media lacking uracil (and, in my case, also lacking
trptophan). The strain is then diluted 100 fold in minimal raffinose
media lacking uracil (and tryptophan) and grown OVERNIGHT at 30 degrees C
with shaking. One then dilutes the culture to an O.D. 595 of 0.3 in the
same minimal raffinose media and galactose is added to a final
concentration of 20g/liter. Incubation at 30 degrees C is continued for
at least 2 hrs. The culture is then said to be "induced."
My problem is that, despite repeated attempts with new batches of media,
the strain does not seem to be growing in raffinose. I'm in the process
of testing the cells on raffinose plates; however, I'm wondering whether
anyone might have an explanation, aside from a mutation loss in the
ability to grow on raffinose, for why the strain fails to grow in raffinose.
Oh, one more thing--My definition of minimal raffinose media is: 6.7 g/L
Yeast nitrogen base without amino acids, 20 g/L raffinose, appropriate
amino acid drop out mix (these mixes have been used many times before, so
I don't suspect them.).
Thanks in advance. You can respond to me directly by email.
--Leon Parker
Department of Microbiology
Mail stop SC-42
University of Washington
Seattle, WA 98195
email: mrclean at u.washington.edu
