USTC/Notebook/Point mutation Quick-Change method

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===The Quick-Change method===
===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.
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.

Latest revision as of 15:10, 29 October 2008

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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.

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