Mutagenesis

William R. Morgan wmorgan at ACS.WOOSTER.EDU
Wed Sep 8 16:11:42 EST 1993


Dave Knecht <KNECHT at UCONNVM.UCONN.EDU> asks for "some advice on which
approach has been successful for others to do site directed mutagenesis".  

In the past, I've used the T7-GEN IN VITRO Mutagenesis Kit from U.S.
Biochemical with success, although I never achieved the mutation frequency
of 75% that they claim.  The method starts with a single-stranded circular
DNA and a mutant oligonucleotide which after annealing is extended  using
T7 DNA polymerase in the presence of 5-methyl-dCTP.  The original,
unmethylated strand is then digested using a methylation-sensitive
restriction enzyme and ExoIII; followed by transformation into an mcrAB-
strain.

The other possibility is to do site directed mutagenesis by PCR which has
been described in various places, including "PCR Protocols" (see the FAQ
for this newsgroup).


Good luck,
Bill Morgan

William R. Morgan
The College of Wooster
Department of Biology
931 College St.
Wooster, OH  44691
Phone:  216-263-2026
FAX:    216-263-2378
E-mail: wmorgan at acs.wooster.edu




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