Problems with Kunkel mutagenesis

Anthony J. Persechini ajp2o at crocus.medicine.rochester.edu
Thu Dec 22 09:30:04 EST 1994


Dear Colleagues:

We are using mismatched primer-extension mutagenesis to make mutations in
both phagemid and M13 DNA. We are using the Sambrook procedure for the
reactions. We are using 1 pmol of template and 20 pmol of primer in our second
strand sytheses; we transform with 1 and 5 ul of the completed reaction.

PROBLEM:
We often find we get more plaques/colonies (sometimes many more) with the
un-primed
control reaction than with the primed reaction. This true with T7 pol and with
Klenow. In spite of this, in some cases we have evaluated the primed reactions
and see good mutation efficiencies. Electrophoresis of DNA from the reactions
sometimes (but not always) shows evidence of significant self-priming, although
we always see MORE successful
second strand synthesis when primer is included.

It looks as though something is inhibiting transformation. Could it be the
primers?

If anyone has seen this sort of thing or has any notion of what might be going
wrong please e-mail
a reply. I will post a summary if I figure this thing out.

-- 
Anthony Persechini
Assistant Professor
University of Rochester School of Medicine
ajp2o at crocus.medicine.rochester.edu



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