USTC/Notebook/Point mutation Quick-Change method

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(Difference between revisions)
(New page: ===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...)
(The Quick-Change method)
Line 4: Line 4:
  system:          concentration        volume
  system:          concentration        volume
     5*PS Buffer    5*                  4.0ul
     5*PS Buffer    5*                  4.0ul
-
     dNTP mixture 2.5mM each          2.0ul
+
     dNTP mixture   2.5mM each          2.0ul
-
     primer1       25uM               0.2ul
+
     primer1       25uM               0.2ul
-
     primer2       25uM               0.2ul
+
     primer2       25uM               0.2ul
-
     template     changeable            ~
+
     template       changeable            ~
-
     PrimeSTAR      ~                0.2ul
+
     PrimeSTAR      2.5U/ul            0.2ul
-
     ddH2O                           add to 20ul  
+
     ddH2O           ~              add to 20ul  
process  program  
process  program  
-
  Predenaturing      94℃       5 min          
+
  Predenaturing      94℃               5 min          
-
   Denaturing     94℃       30sec  
+
   Denaturing     94℃               30sec  
-
   Annealing   follow the Tm 30sec                   30cycles
+
   Annealing   follow the Tm         30sec             30cycles
-
   Extension     72℃       theoretically 1kb/min
+
   Extension     72℃         theoretically 1kb/min
-
   Last extention       72℃       10min
+
   Last extention     72℃               10min
-
   Hold             10℃
+
   Hold               10℃
DpnI digestion of the amplification products: DpnI will digest parental methylated and hemimethylated DNA .
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.
Transformation: follow the standard protocol. Nick of the mutated molecule will be repaired in cells.

Revision as of 09:09, 29 October 2008

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.