mw132 at mole.bio.cam.ac.uk
Wed Sep 5 12:40:29 EST 2001
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
> Hope this helps,
> > 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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