site directed mutagenesis

Vladimir Svetlov svetlov at oncology.wisc.edu
Wed Nov 11 17:22:42 EST 1998


In article <36496A6C.B9345561 at lg.ehu.es>, Oskar Fdz-Capetillo
<ggbferuo at lg.ehu.es> wrote:

> Hi there:
>     I am just starting to develop my mutagenesis techniques. I am
> wondering about using the "quick change" Kit from Stratagene. I would
> want to know if anybody has used this kit and how did it work.
>     I would like to know any other site mutagenesis protocols if you
> have tried them succesfully.
>     Hoping to hear about you soon and thank you in advance,
>                 Ana Aguirre

The QuickChange kit from Stratagene works very efficiently (90%+ for point
mutants, 50%+ for deletions of up to 1 kb and insertions of up to 100 bp).
As there were some backorders with the kit we were forced to discover (we
would never think of not buying the entire kit on our own...) that the
method works fine with Pfu purchased separately from Stratagene or Promega
and DpnI from just about anybody. Once I've used Pwo instead of Pfu and
that worked too. The largest plasmid I ever used with this protocol was 7
kb long. Let me know if you have specific questions about particular type
of mutations you want to introduce. Overall this is by far the easiest and
most efficient SD mutagenesis kit we have used.

Regards,
V.

-- 
Vladimir Svetlov, PhD
McArdle Lab for Cancer Res.
UW-Madison
1400 University Ave.
Madison, WI 53706



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