With regard to the 2 recent questions:
1. We have successfully done PCR from various yeast extracts. As with all
PCR, conditions must be worked out carefully for each Primer/Template set,
especially temperatures and Mg concentration. We find shortening the 94 C time
to as little 30 sec for the first cycle and 10 sec for subsequent cycles may
improve backgrounds and yields. Of course, avoid contamination.
2. All yeast plasmid constructs are unstable. You should only maintain
strains under selective conditions, or the majority of cells (in most cases the
vast majority) will lose the plasmids. We never grow any plasmid containing
yeast cell under non-selective conditions unless we wish to eliminate a plasmid
(which we frequently do, and never fails).
Sincerely,
Mike Leibowitz
UMDNJ-RW Johnson Medical School
