Team:SUSTC-Shenzhen-B/protocol
From 2012.igem.org
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
Restriction Enzyme Digestion for Verification
After the PCR mutation we have to do a restriction enzyme digestion to test whether the mutation is successful. The reaction system is as follows:
1. Prepare the control reaction as indicated below:
Total: 10μl
+ 0.5μl of Pst I restriction enzyme
+ 1μl of 10XH buffer
+ 1μl of plasmid DNA
+ 7.5μl of ddH2O
2. Prepare the sample reaction as indicated below:
Total: 25μl
+ 0.5μl of Afl II restriction enzyme, #ER0831
+ 1μl of 10XM buffer
+ 1μl of plasmid DNA
+ 1.0μl of 0.1% BSA
+ 6.5μl of ddH2O
After the reaction, do an electrophoresis to see the 2k bp cyclic plasmid DNA change into linear. The process is as follows: