Team:NCTU Formosa/protocol

Protocols

Point Mutation

The procedure is as follows :

1. Design primers
2. Find the best PCR condition by gradient PCR
   
3. 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 25

   94℃ 5 min 94℃ 30 sec 55℃ 30 sec 72℃ 5 min 72℃ 7~10 min Cycles : 25

4. Confirm the PCR product with electrophoresis
5. DPN1 37℃ for 3hr~overnight
   

   DPN1 0.5 Buffer2 2 PCR product 17.5 Total 20

6. 80℃ 20mins to denature the DPN1
7. Confirm with electrophoresis
8. Self ligation room temperature for 2~3 hr
   

   Enzyme 1 Buffer 2 ATP 2 H2O 5 PCR product 10 Total 20

9. Transform DH5alpha with the self-ligation product
   

   Self-ligation product 20 DH5alfa 50

10. Incubate in Ap25 plate then transfer to Ap50 plate