Overlap Extension PCR

Overlap Extension PCR is described in Molecular Cloning (Sambrook). Basically involves designing two mutagenic primers, containing the mutation and partially or completely complementary to each other. (I make them entirely complementary) Each primer is used in a separate reaction (separate tubes, same conditions) with an outer flanking primer designed to one end of the region of interest. You generate two halves of the region in this manner in two separate reactions and put them together in the next step, where they anneal in the 25-30 bp region of complementarity and prime off each other, to give you the full length product. All you need is Pfu polymerase, and the target sequence on a plasmid. I make sure that the Tms of the four primers are in the same range, and bump up the annealing temperature for the second PCR.