Site directed mutagenesis

Ashok Aiyar axa12 at
Thu Oct 29 19:30:22 EST 1992

In article <1992Oct27.175409.26280 at> PHILLIPSA at UK.AC.AFRC.LARS (Andy Phillips) writes:

>Dear PCRologists and homebrewers,
>I need to do some mutagenesis on a 500 bp stretch of DNA, to
>create restriction sites about every 100 bp. I haven't done
>any multiple site-directed mutagenesis since the dark ages B.P. 
>(Before PCR) and haven't kept track of the methodology papers.
>For cost reasons, I would prefer to make only one mutagenic
>primer per site. Has anyone out there tried (with success) the
>"megaprimer" method (Sarkar & Sommer BioTechniques (1990) 8:404-7)?
>Or do you know a better method?

All the time.  It works has worked very well for us in a region of Rous 
Sarcoma Virus that has been recalcitrant to mutagenesis using single-
stranded DNA.  We have a small modification of the method that will be 
published in the April '93 issue of BioTechniques, that increases the yield 
of the final product several fold.

                             Ashok Aiyar
                     Department of Biochemistry
                         CWRU Medical School
                          axa12 at

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