'Tandem' Primers for PCR Mutagenesis

Andy amcshea at world.std.com
Fri Apr 8 18:41:26 EST 1994


Shaun D. Black (SHAUN at JASON.UTHCT.EDU) wrote:
: Howdy All,

I agree with Steve Rodems, although I found that sometimes I had to include a  
fourth reaction.
My approach:
1) Produce LH fragment by PCR
2) Produce RH fragment by PCR with the 5' primer containing the desired
mutation.

RH 3' primer and LH 5' primer had an 8bp overlap, note that the RH 3' 
primer DID not bind the mutation region in my case.

3) Gel purify the LH and RH fragments. Set up a PCR without primer 
without primers for say 15 cycles to amplify the larger fragment LH
frag+RH frag. and use this as a template for reaction 4.
4) Final PCR using outermost primers.
Gel purified the desired band, digested to produce the mutated fragment
with suitable ends that were within the amplified sequence cloned and
sequenced.
        Worked nicely but had a few mutations swapped base or two and a
frameshift at the mutated primer binding site in some of the clones.

        All the Best,


                                Andy 




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