Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

mutation using overlapping PCR, what's the success rate? - (Mar/14/2011 )

Hi,
I was given a suggestion of using overlapping PCR to do a single nucleotide mutation, but some other people reckons that it's not going to work with a reason that the primers will mostly bind to the amplified portions and to get a full long product (overlapping)with a single nucleotide mutation in the middle of it will be almost impossible. Just wondering if anyone out there has any inputs with this method, and any suggestion with protocols that i can use?
thanks in advance!

-wonky-

You can use Dpn1 treatment after your 1st PCR to digest old plasmid specifically. Usually the sucess rate is fairly high, maybe 95%.

-Rsm-

didn't really work well for me, as I found out <_<

-wonky-

wonky on Wed Jun 29 05:51:11 2011 said:


didn't really work well for me, as I found out <_<

Could you tell us your protocol?

-Rsm-