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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.
TheDpn 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.