- The water bath was set at 42 ℃, and Heat block was set 37 ℃.
- Add 280 μL of LB medium to an empty microtube and place it in the heat block.
- Plasmid about 1 or 2 μL was added to the microtube containing 10 μL of competent cells and stirred by tapping.
- Left on ice for 15 minutes.
- Heat shocked on water bath for 45 seconds.
- And the microtube containing the plasmid is quickly returned to ice and left still for 2 minutes.
- And 250 μL of LB medium is added.
- Incubate at 37 ℃ for 1 hour
- 20 μL of chloramphenicol was applied to LB agar medium.
- After culturing, 125 μL of the solution was applied to the agar medium, and cultured overnight at 37 ℃.
Restriction enzyme treatment
- Heat block was set at 37 ℃.
- Prepare new microtube.
- Add 1 μL of ×10 restriction enzyme buffer to the microtube.
- Next, 1 μL of DNA sample is added.
- And, 1 μL of restriction enzyme is added.
- D2W was added so that the total contents of the microtube becomes 10 μL.
- The microtube was then placed in heat block set at 37 ℃ and left reacted for more than 1 hour.
- A heat block is set at 16 ℃.
- Prepare a new 1.5mL microcentrifuge tube.
- Add PCR products to the 1.5mL microcentrifuge tube.
- Add the plasmid vector after the treatment of restriction enzyme to the 1.5mL microcentrifuge tube.
- Transfer 2x Rapid Ligation Buffer into the 1.5mL microcentrifuge tube.
- Add T4 DNA Ligase into the 1.5mL microcentrifuge tube.
- Then add Nuclease free water into the 1.5mL microcentrifuge tube.
- This 1.5mL microcentrifuge tube will then put in a heat block and incubate for 20 minutes at 16℃
DNA purification was performed using FAVORGEN according to the respective manufacturer's protocol.