USTC/Notebook/Point mutation Quick-Change method

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The Quick-Change method
Primer design: both of the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid. The desired mutation should be in the middle of the primer with ~10-15bases of correct sequence on both sides. On each side of the desired mutations of my primers, 4*(the number of Gor C)+2*(the number of A or T)>45. It is better that the primers terminate in one or more C or G bases. Polymerase Chain Reaction: we use PrimeSTAR (TaKaRa) as the polymerase. system:         concentration         volume 5*PS Buffer   5*                  4.0ul dNTP mixture  2.5mM each          2.0ul primer1       25uM                0.2ul primer2       25uM                0.2ul template      changeable             ~ PrimeSTAR     2.5U/ul             0.2ul ddH2O           ~               add to 20ul process program Predenaturing      94℃                5 min Denaturing	   94℃                30sec Annealing	  follow the Tm        30sec	            30cycles Extension	   72℃         theoretically 1kb/min Last extention    72℃                10min Hold              10℃

DpnI digestion of the amplification products: DpnI will digest parental methylated and hemimethylated DNA.

Transformation: follow the standard protocol. Nick of the mutated molecule will be repaired in cells.