Team:SUSTC-Shenzhen-B/protocol

Lab Protocol

brief process

1. Site-Directed Mutagenesis

2. Restriction enzyme digestion and electrophoresis (proof)

3. Media Preparation

4. Bacterial Transformation

5. Colony PCR for verification

6. Culture the bacteria

7. Plasmid DNA Isolation

8. Restriction Enzyme Digestion and Electrophoresis(proof)

9. Amplify GFP & RFP

10. Electrophoresis and Restriction Enzyme Digestion.

11. Ligation

12. Bacterial Transformation

13. bacterial colony PCR for verification

14. Culture the bacteria

15. Plasmid DNA Isolation

16. Restriction Enzyme Digestion and Electrophoresis

Site-Directed Mutagenesis

We choose plasmid psb1a3 to be the vector that ligate GPF and RFP fragments.To protect the structural integrity of the constructed plasmid, we need to mutate a restriction enzyme cutting site named Pst I to Afl II. Proper primer are designed for this purpose.

PtoA-F:5'-CCACCTGACGTCTAAGAAAC-3'

PtoA-R:5'-ATGATCATCGCCGGCGAATTCAGGC-3'

1. Prepare the sample reaction as indicated below:

Total: 25μl

+ 0.25 μl of Ex Taq polymerase(酶的公司名称)

+ 2.5 μl of 10× Taq reaction buffer

+ 2.0 μl of dNTP(2mM)

+ 1.0 μl of template

+ 1.0 μl of oligonucleotide primer P1

+ 1.0 μl of oligonucleotide primer P2

+ 18.25 μl of ddH2O

2. Set thermocycler temperatures and the time. Procedure on the thermocycler are listed below:

① 94˚C for 5 min

② 30 cycle

a. 94˚C for 1 min

b. 55˚C for 1 min

c. 72˚C for 1 min20sec

③ 72˚C for 10 min

④ 4˚C for stock