Quick Change mutli kit
Laura.Segall at mail.mcgill.ca
Mon Dec 1 14:59:23 EST 2003
I was wondering if anyone can help me out.
I am trying to introduce several mutations into a plasmid (6Kb) using
the stratagene quick change multi-site kit. All primers were designed
as suggested in the kit manual, but I obtained no PCR product. Template
was checked on a gel prior to PCR, primers were phosphorylated, cleaned
on a column and quantified. Still no product. Annealing temp was
dropped to 52C (from 55C), still no product. Primer concentration was
fiddled with, still no product. Technical assistance suggested
transforming the bugs anyway, saying that the PCR yield may simply be
too small to see on a gel. Personnally, I prefer to see a product on a
gel first. Other than that, they had no suggestions. I am trying the
transformation tonight and will fiddle with the ratios of template:primer.
Any help/suggestions would greatlty be apreciated.Getting a little
thank you in advance,
Laura Segal, Ph.D.
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