Cloning by Overlap PCR

Mad Mat hauwelm at
Tue Apr 20 06:55:14 EST 2004

NRohitlall at ("Neeresh Rohitlall") wrote in message news:<s083a0e7.016 at>...
> Hello,
>  does anyone have a good protocol for mutagenesis using Overlap PCR?The
> idea is to PCR the gene from the mutation site (forward and reverse from
> the middle of the gene where mutation is to introduced) and then use the
> the sequencing primers to pcr the whole gene which should include the
> inserted mutaion. Does this make sense?am new to PCR strategies.
> many thanks in advance
> N Rohitlall
> South Africa

You got the idea. You have to generate two fragments of your gene and
anneal-extend them to reconstitute the whole gene. The two fragment
should overlapp on a decent length (at least 20 bp). The first part is
a standard PCR with one primer that introduces the mutation. Then mix
the two gel-purified fragments together and do 7 cycles of
94 C 30s
40 C 45s ramp 30C/min (this temperature depends on the Tm of the
70 C 1m
Only then add the primers (your so-called sequencing primers) and do
30 cycles of
94 C 30s
55 C 30s
70 C 1m

This protocol worked fine with me for many applications, it just
requires a bit of optimisation for the annealing t.

Good luck


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