Team:KAIT JAPAN/notebook/protocols

Protocols

Transformation

1. The water bath was sat at 42 ℃, and Heat block was sat 37 ℃.
2. Add 280 μL of LB medium to an empty micro tube and place it in the heat block.
3. Plasmid 1 or 2 μL was added to the microtube containing 10 μL of competent cells and stirred by tapping.
4. Left on ice for 15 minutes.
5. Heat shocked on water bath for 45 seconds.
*1 Punctuality
6. It quickly returned to ice and left to stand for 2 minutes.
7. Added 250 μL of LB medium.
8. Incubate at 37 ° C for 1 hour
9. 20 μL of chloramphenicol was applied to LB agar medium.
10. After culturing, 125 μL of the solution was applied to the agar medium which was subjected to 9, and overnighted at 37 ° C.

Restriction enzyme treatment

1. Heat block was set at 37 ° C.
*1 Since it depends on each restriction enzyme, refer to the manual.
2. Prepared one microtube.
3. Added 1 μL of ×10 restriction enzyme buffer to the microtube.
4. Next, 1 μL of DNA sample is added.
5. Next, 1 μL of restriction enzyme is added.
6. D2W was added so that the total content of the microtube was 10 μL.
7. The microtube was placed in a heat block set at 37 ° C and reacted for 1 hour or more.

Ligation

1. A heat block is set in 16 ℃.
2. Prepare the 1.5mL microcentrifuge tube.
3. Add PCR products to the 1.5mL microcentrifuge tube.
4. Add the plasmid vector which did restrict enzyme treatment to the 1.5mL microcentrifuge tube.
5. Transfer 2x Rapid Ligation Buffer into the 1.5mL microcentrifuge tube.
6. Transfer T4 DNA Ligase into the 1.5mL microcentrifuge tube.
7. Transfer Nuclease free water into the 1.5mL microcentrifuge tube.
8. This 1.5mL microcentrifuge tube put in a heat block and incubate for 20 minutes at 16℃

PCR

1. Add D2W to PCR tube.
2. Add MgCl2 to PCR tube.
3. Add 5x Colourless Buffer to PCR tube.
4. Add dNTP to PCR tube.
5. Add VF2 to PCR tube.
6. Add VR to PCR tube.
7. Add Taq polymerase to PCR tube.
8. Add sample to PCR tube.
9. Make RCR tube puted in the thermal cycler.
10. Start the PCR reaction.