HiPer® PCR Mediated Site Directed Mutagenesis Teaching Kit

The site-directed mutagenesis method is performed using Pfu Ultra high-fidelity DNA polymerase for mutagenic primer-directed replication of both plasmid strands. The basic procedure utilizes a supercoiled double-stranded DNA (dsDNA) vector with an insert of interest and two synthetic oligonucleotide primers, both containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by Himedia’s Hi-Proof DNA polymerase, without primer displacement. Extension of the oligonucleotide primers generate a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I. The Dpn I endonuclease (target sequence: 5´-Gm6ATC-3´) is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for mutation-containing synthesized DNA. DNA isolated from almost all E. coli strains is dam methylated and therefore susceptible to Dpn I digestion. The nicked vector DNA containing the desired mutation is then transformed into XL1-Blue competent cells.