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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. | + | 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. | Polymerase Chain Reaction: we use PrimeSTAR (TaKaRa) as the polymerase. | ||
system: concentration volume | system: concentration volume | ||
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process program | process program | ||
Predenaturing 94℃ 5 min | Predenaturing 94℃ 5 min | ||
| - | Denaturing | + | Denaturing 94℃ 30sec |
| - | Annealing follow the Tm | + | Annealing follow the Tm 30sec 30cycles |
| - | Extension | + | Extension 72℃ theoretically 1kb/min |
Last extention 72℃ 10min | Last extention 72℃ 10min | ||
Hold 10℃ | Hold 10℃ | ||
Latest revision as of 15:10, 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.
