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Team:NCTU Formosa/protocol
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Revision as of 16:15, 4 October 2011
Protocols
Point Mutation
The procedure is as follows :
- Design primers
- Find the best PCR condition by gradient PCR
- KOD PCR condition
Plasmid 0.5 μl
pF 0.5
pR 0.5
MgCl2 1
dNTP 2.5
buffer 2.5
KOD enzyme 0.5
H2O 17
Total 2594℃ 5 min
94℃ 30 sec
55℃ 30 sec
72℃ 5 min
72℃ 7~10 min
Cycles : 25 - Confirm the PCR product with electrophoresis
- DPN1 37℃ for 3hr~overnight
DPN1 0.5
Buffer2 2
PCR product 17.5
Total 20
- 80℃ 20mins to denature the DPN1
- Confirm with electrophoresis
- Self ligation room temperature for 2~3 hr
Enzyme 1
Buffer 2
ATP 2
H2O 5
PCR product 10
Total 20
- Transform DH5alpha with the self-ligation product
Self-ligation product 20
DH5alfa 50
- Incubate in Ap25 plate then transfer to Ap50 plate
