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 theexceptional 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 andthe parental plasmid.

Following temperature cycling, the product is treated with Dpn I.TheDpn I endonuclease (target sequence: 5´-Gm6ATC-3´) is specific for methylatedand hemi-methylated DNA and is used to digest the parentalDNA template and to selectfor mutation-containing synthesized DNA.

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