Dear yeast netters,
A couple of weeks ago I posted a request for PCR protocols on whole yeast
cells. I want to thank you for your numerous replies. It's clear from the
varied nature of the replies that many people out there are working too
hard to do PCRs, at least for non-critical applications.
Let me summarize our current experience:
1. As many of you have said, THERE IS NO NEED TO ISOLATE DNA. We have
found that one toothpick's worth (we use the small end of a disposable
innoculating loop to avoid contamination), or about 1/2 of a 2 day YPD
colony, resuspended directly in a 100 microliter PCR reaction, works just
fine. There is also no need to preboil; just resuspend and go.
2. We have also found that the PCR product is cuttable by at least one
restriction enzyme (Sal1) DIRECTLY from the tube. Kind of surprising with
all those yeast guts swimming around, but there you have it. Just spin the
tube to pellet the glop and use the supernatant.
Needless to say, with this kind of ease of use, whole new worlds have been
opened up to us in terms of genetic screens. What a great technique. The
guy who invented this should get some sort of prize, at least.
Thanks to all who helped,
lichten at helix.nih.gov