Site directed mutagenesis

Dennis J. Templeton djt2 at po.CWRU.Edu
Wed Oct 28 12:48:53 EST 1992


In a previous article, PHILLIPSA at UK.AC.AFRC.LARS (Andy Phillips) says:

>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?
>

We have used the "megaprimer" approach with great success... in fact we 
*thought* we had invented it and then found the reference cited above after 
the fact.  

I still worry about umwanted mutations creeping in through PCR, and 
would suggest subcloning back a small piece that you sequence in its
entirety.

good luck, 
dennis



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