Team:KAIT JAPAN/methods

Experimental Method

Transformation

  1. The water bath was set at 42 ℃, and Heat block was set 37 ℃.
  2. Add 280 μL of LB medium to an empty microtube and place it in the heat block.
  3. Plasmid about 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]
  6. And the microtube containing the plasmid is quickly returned to ice and left still for 2 minutes.
  7. And 250 μL of LB medium is added.
  8. Incubate at 37 ℃ 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, and cultured overnight at 37 ℃.

Restriction enzyme treatment

  1. Heat block was set at 37 ℃.[2]
  2. Prepare new microtube.
  3. Add 1 μL of ×10 restriction enzyme buffer to the microtube.
  4. Next, 1 μL of DNA sample is added.
  5. And, 1 μL of restriction enzyme is added.
  6. D2W was added so that the total contents of the microtube becomes 10 μL.
  7. The microtube was then placed in heat block set at 37 ℃ and left reacted for more than 1 hour.

Ligation

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

DNA purification

DNA purification was performed using FAVORGEN according to the respective manufacturer's protocol.