Team:BEAS China/Protocol
Cloning
PCR
Reaction system:
Process:
Gel Purification
We used the gel extraction kit(TIANGEN, DP209-02) to get the objective fragment.
Gibson Assembly
Reaction system:
Process:
Transformation
1. Preparation of the competent cells.
2. Ligation product + 50 μL competent cells
3. Heatshock in 42°C for 90s
4. Incube on ice for 2min
5. Add 600 μL LB media and incubate for 1h(37°C, 220rpm)
6. Centrifuge at 4000 rpm for 1min and remove 400 μL supernatant
7. Resuspend the pellets and use 100 μL supernatant to spread plates(with antibiotic)
8. Incubate for 12~16h(37°C)
Protein purification
We used the His-tag Protein Purification Kit(Beyotime P2226) to purify protein.
We tested the purified protein with SDS-PAGE.
SDS-PAGE gel recipe
1. After ensuring that the equipment is waterproof, the running gel is mixed and filled into the chamber. Pipetting about 1ml of H2O on top of the running gel to seal the gel.
2. After polymerization, the remaining H2O is removed and the stacking gel is filled on top. Insert a comb to create sample pockets.
3. After the stacking gel also polymerized, 1xrunning buffer is used to run the Double Gel System via the SDS gel.
4. After loading the generated pockets with the samples, the stacking gel is run at 100 V and then running gel at 120V.