Polymerase Chain Reaction (PCR) is a method of making multiple copies of a DNA sequence, involving repeated reactions with a polymerase enzyme, which comes in different types, such as Taq-polymerase, Phusion or Q5. To run a PCR reaction, one needs a DNA template to be amplified, one set of forward and reverse primers, a polymerase enzyme as well as various buffers. This protocol describes the procedure to perform a two-step PCR reaction, in which the primers contain an extra non-binding overlap sequence which allows the assembly of a new sequence from a specific region of the template DNA. This is done by using two sets of primers, one which adds a non-binding overlap sequence and one other set which add a desired sequence binding to the overlap region introduced by the first set of primers.
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