>> >Look in the November isuue if Biotechniques. There is a simple method that
> >does not involve plasmid isolation prior to PCR.
>> I've PCR'ed genomic targets from S. pombe and S. cerevisiae by the
> simple expedient of picking a small amount of cells from a colony and
> twirling it in the PCR reaction mix. I can't imagine it wouldn't work
> from plasmids in yeast as well.
Yup. Works great. I've been doing just that on 2-hybrid clones for several
I just pick up a one or two microliter-sized glop of cells from a patch or
colony with a pipette tip and touch it to the bottom of a clean PCR tube,
leaving behind a small glop (about 1 ul) of cells. Then I add pre-mixed
PCR cocktail to all my tubes and go cycle (don't even need to mix). Too
many cells is definitely inhibitory. Better still if you have a fancy
thermocycler that uses thin-walled tubes. I get 30 cycles done in 90
minutes, then cut 1 ul of my 25ul reaction with restriction enzymes and
run onto a gel.
I have found that the age of the colonies is important. I'm picking cells
from the 2-hybrid XGal indicator plates, and if the plates are too old, or
been in the fridge for a while, then >15% of the reactions fail and the
yield goes down dramatically. Just today I PCR'ed some cells which have
been resting on my benchtop after growth for about one week - they were
still OK. Good luck.
cfritze at cts.com