multi-site mutagenesis

[Bart Frank]

I am looking for an efficient method to mutate 3-4 different
sites at the same time within a sequence. Any recommendations???
Thanks,
Baer

[Bob's lab]

If the sites you want to mutate can be designed on a single primer then
have a look at the Quikchange protocol (from Stratagene). The results
are very good, at least for us, but it is fairly expensive. If you have
a look at the protocol, you may not need the kit (it's basically a PCR
kit with additional enzymes for digesting the template DNA, and relies
on two complementary mutagenic primers to introduce the mutation). The
super-competent cells they refer to can be substituted for a strain
such as JM101, or if you have a mutant strain for your gene of
interest, then use that instead (very handy for complementation
analysis).

Good luck,

Stuart (No connections to Sratagene!!!!)

[zhu32...@gmail.com]

I think type 2 restriction enzyme (like BsmB1)-depentdent site-directed mutagenesis can solve your problem. You can finished mainly process in a day.