mutagenesis kit

Michael Witty mw132 at mole.bio.cam.ac.uk
Wed Sep 5 12:40:29 EST 2001


Dear Robert,
            do you mean you PCR around the circumerence of the plasmid,
using the mutagenic primer pair and then transform with linear DNA?  Or do
you cut and ligate?

I have been using a kind of two stage PCR reaction using 4 primers, two
of which overlap:

---->             <----
                   ---->              <----

. . . if you see what I mean.  Regards, Mike.


On 5 Sep 2001, Robert J. Nelson wrote:

> Hi there,
>
> The Stratagene Quikchange PCR-based method has been extensively in our lab
> for site-directed mutagenesis with great success.  We use their method, but
> don't buy the kit because it's quite expensive.
>
> All you need to do is a PCR with Pfu and complementary oligos spanning your
> mutagenic site, Dpn I digest away the methylated template DNA, then
> transform your bugs (you don't even need fancy ones that Stratagene suggest
> - standard lab host strains can handle the nick repair).  Pick clones the
> following day, miniprep and hey presto!
>
> In my hands I get about 50 - 75% of transformants with the mutation
> correctly incorporated (analysed by sequencing), the other 25 - 50% being
> the original template and occasionally a plasmid with the mutagenic region
> duplicated.
>
> Hope this helps,
>
> bob
>
>
> > Dear all:
> >
> > It has been a while since I used the promega M13-based mutagenesis kit
> > back in 1994. Does anyone use more advanced commercial kit for in vitro
> > mutagenesis? I found Clonetech " Transformer" and Strategene
> > "Quickchange" kits. Welcome any users opinion on these products. Thanks.
>
> ---
>




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