Take any plasmid in which the gene of interest is inserted.

Multiply this plasmid within a methylating bacteria.

( While plasmid DNA isolated from almost all of the commonly usedE. coli strains (dam+) is methylated and is a suitable template formutagenesis, plasmid DNA isolated from the exceptional dam –E. coli strains, including JM110 and SCS110, is not suitable)

Order a pair of primer with the mutation you want to introduce ( x ) in a thermocycler, denature the plasmid.

Anneal the primers.

Extend the primers with a Pfu DNA pol.

Extension of the oligonucleotide primers generates a mutated plasmid containing nicks and the parental plasmid.

Following temperature cycling, the product is treated with Dpn I.

The Dpn I endonuclease (target sequence: 5 ´-Gm6ATC-3´) is specific for methylated and hemi-methylated DNA and is used to digest the parentalDNA template and to select for mutation-containing synthesized DNA.

The nicked vector DNA containing the desired mutations is purified and then transformed into competent cells.