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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.