plasmid rescue from yeast

Nancy Lemieux nhl at binkley.cs.mcgill.ca
Tue Mar 15 13:43:09 EST 1994


Yes, I have to agree totally with Fatima, that plasmid rescue for Southerns
is very tricky.  In fact, I did one of those less than a month ago.  There
are several tricks that you can try.  I started by doing a plasmid extract
with acid washed glass beads as described in the red book, vol 2.  I  then
precipitated with isopropanol (which they say is dangerous because it
concentrates inhibitors), kept an aliquot, and divided the rest into two
experiments.  For one half, I did a phenol, a phenol/chloroform, and then a
chloroform extraction.  For the other half, I just did a regular GeneClean.
 I transformed approximately equal amounts of each plasmid into CaCl2
competent E.Coli.   I got zip amount of colonies which meant one of two
things: 1. There is indeed an inhibitor ; or 2. The cells weren't competent
enough.

I found a paper, I can't remember the reference right now, where they
checked for inhibition by cotransforming the yeast isolated plasmid with
another plasmid (e.g. bluescript, pBR322, pGEX, etc).  I tried this by
cotransfecting my plasmid with pGEX-2T and found that I had equal amounts
of transformants on all the plates (i.e. pGEX-2T with any yeast plasmid
prep, or even just pGEX alone).  This ruled out inhibition and meant my
cells were not competent enough.   

I then made a batch of electrocompetent cells and transformed my
isopropanol prep with an aliquot and got colonies which gave the right
restriction digest and lit up as my plasmid with the right insert on a
Southern.

The moral of my story is that I no longer believe as much in inhibitors as
I do in competency.  My CaCl2 cells gave approx. 1X10^5 transformants per
microgram of DNA, while my electrocompetent ones gave 10^8 to 10^9
transformants.  As further proof, another member in our department had the
same problem, and solved it by electroporating.

Good luck!


Nancy Lemieux
Dept. of Biochemistry
McGill University
Montreal, Canada
nhl at binkley.cs.mcgill.ca



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