Yeast PCR Advice

Christian Fritze cfritze at cts.com
Wed Nov 29 19:24:41 EST 1995


> 
> >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
weeks now. 

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.

-- 
Christian Fritze
cfritze at cts.com



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