Site directed mutagenesis

Ashok Aiyar axa12 at po.cwru.edu
Thu Oct 29 19:30:22 EST 1992


In article <1992Oct27.175409.26280 at gserv1.dl.ac.uk> 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
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                             Ashok Aiyar
                     Department of Biochemistry
                         CWRU Medical School
                          axa12 at po.cwru.edu
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