Stratagene QuikChange--tandem inserts!!!

Dave Smith davesmith at bioch.tamu.edu
Wed Nov 13 17:30:12 EST 1996


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're baffled......and so was the Stratagene rep who said he'd never
heard of such of a thing and wasn't going to bother the original
researchers with the same.

Anyone else see this sort of result??  We can rationalize a dimer
tandem insert in situations where primer is limiting.  In this case
it's probably the Pfu polymerase extending the annealed products from
a previous round of amplification to completion without having to
displace any competing primer.  But for fivemers, we just can't
envision a model.

Thanks for any input and advice!
Dave Smith
Ry Young's Lab
Dept. of Biochemistry and Biophysics
TAMU




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