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.