PCR on whole yeast cells

Michael Lichten lichten%bchem.dnet at DXI.NIH.GOV
Fri Oct 15 07:15:45 EST 1993


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,

Michael Lichten
lichten at helix.nih.gov



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