Invitro mutagenesis

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Mar 29 10:29:12 EST 2001


When I was a graduate student, I did a lot of mutagenesis via the Kunkle
method.  It was generally very successful, but I did have one mutant I
wanted to make that just never worked with that method.  In the end, I used
a PCR-based approach.  I'm sure you could find such methodologies described
in detail in something like "Current Protocols in Molecular Biology" or
even someplace on the web.  I've had very good success using PCR to do site
directed mutagenesis.  In fact, this is about the only way I do it now.

Mike


>Hi,
>I have a problem. I have cloned a 1.4kb gene into BamHI site of pSBETa
>vector,
>I have been trying to do invitro mutagenesis using quickchange mutagenesis
>kit
>from stratagene, somehow its not working with my plasmid, but it works with
>the control plasmid provided along with the kit. I have tried various
>concentrations of both template as well as primers.
>Could anyone suggest some solutions to this problem, I would be very greatful
>if anyone suggest me some solutions which could solve my problem.
>
>Thanking you
>
>Dinesh
>Dept. of Botany
>The University of HongKong
>Hongkong
>andinesh at hkucc.hku.hk
>
>---


Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax

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