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mutation using overlapping PCR, what's the success rate?


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#1 wonky

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Posted 14 March 2011 - 09:24 PM

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!

#2 Rsm

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Posted 15 March 2011 - 12:21 AM

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

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Posted 28 June 2011 - 09:51 PM

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

#4 Rsm

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Posted 28 June 2011 - 10:27 PM

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

Could you tell us your protocol?
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