Stratagene QuikChange--tandem inserts!!!

William Rice william.rice at utoronto.ca
Sun Nov 17 10:14:29 EST 1996


In article <56db9u$hk5 at news.tamu.edu>, davesmith at bioch.tamu.edu (Dave
Smith) wrote:

> Our lab has used Stratagene's QuikChange kit several times  (3
> separate sets of primers all with different plasmid templates).
> Recently, on the fourth go around, with a new set of primers and new
> template we got some inexplicable results.  Thought we might let
> others know of the possible "hazards" and possibly get some thoughts
> from the mol.bio. community.  We sequenced five candidates. Two were
> parentals.  Of the other three, two had tandem insertions of the
> mutagenic primer at the correct location.  The other had a nine base
> deletion in the middle of the 42 base primer sequence.  In the first
> tandem clone there were FIVE copies of the mutagenic primer, all
> missing between 1 and 6 bases at the junctions except for the last
> copy which was perfect.  In the other tandem repeat there were three
> and a half tandem copies of the primer in the correct location.  For
> those unfamiliar with the kit, there is NO LIGATION step in the
> protocol.  You just PCR, cut with DpnI to "eliminate" template, and
> transform.

We've been using the kit in our lab for several months now, and I've seen
the same thing happen on occasion. Once, when I was sequencing several
positives I could see a "ladder" of insertions, ranging from between 2 and
5. It seems to be primer-specific, since this only happened on a few
primers. The most troublesome primer had the 3' half nearly identical to
the 5' half. We've switched to using a higher annealing temperature than
55C, or even skipping that step altogether for primers with a very high
melting temperature.

Bill

-- 
William Rice
C.H Best Institute, University of Toronto
william.rice at utoronto.ca

....Still waiting for EDLIN '95



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