How to prepare Yeast for PCR?

Kevin A. Morano kevin.a.morano at uth.tmc.edu
Mon Mar 12 15:08:05 EST 2001


...Could anybody tell me an easy way to perform an RAPD on S.
cerevisiae?....

If your RAPD  fragment is small, say 1 kb or less, and your PCR robust,
you can do it on whole yeast cells.  Just grow up fresh yeast on a
plate, and swipe a sterile 200 ul plastic pipette tip through the
colony.  Transfer whatever sticks (half a good-sized colony should
work) to a PCR master mix, including Taq.  Use a 5-7 min hold at 95 deg
prior to cycling in your thermocycler, and run for 30 cycles.  The
repeated heat/cool appears to lyse the cells fairly well, and you
should see a product.  Long or weak PCRs don't work, and there are
reports of PCR inhibition by toothpicks, so use the pipette tip.

Otherwise, you can do a very easy smash-and-grab gDNA prep.  Email me
for details.

Kevin Morano
kevin.a.morano at uth.tmc.edu






More information about the Methods mailing list