Team:KAIT Japan/methods

Experimental Method

Confirmation electrophoresis

  1. ①Place 0.20g of agarose (powder) on the electronic balance, ② 1xTE buffer 20mL in a measuring cylinder, ③ put the stirrer bar in the flask.
  2. Dissolve in a hot stirrer until clear and colorless.
  3. Set the solidified gel on the electrophoresis layer so that the wells are near the cathode of the electrophoresis tank and pour 1xTE buffer until the gel is immersed. Prepare a loose film and adjust the target Sample, Dye, and D2W according to the dilution ratio of Dye.
  4. Pipetting is performed on the parafilm, and the entire amount is sucked up and inserted to the wells of the gel.
  5. After applying all the samples and markers, perform electrophoresis.
  6. Wear a glove in one hand.
  7. Remove the gel taken out of the electrophoresis tank from the cassette and put the gel in a container containing ethidium bromide.
  8. Stain for 10minutes.
  9. After 10minutes, place the dyed gel on the wrap prepared in advance and wrap it.
  10. Observe the band with a UV illuminator.

Transformation

  1. Set the water bath to 42℃ and the heat block to 37℃.
  2. dissolve the Competent cell 10μL in ice.
  3. Weigh 280μL of LB medium into a microtube and heat with heat block.
  4. Put 1-2μL of plasmid in a microtube containing competent cells.
  5. place it on ice for 15 minutes.
  6. place in a 42℃ water bath and heat shock for 45 seconds.
  7. Quickly return it to the ice and leave for 2 minutes.
  8. Add 250μL of warmed LB medium.
  9. 37℃, 1 hour culture time.
  10. During culture, apply 20μL of ampicillin (100mg/mL) and 20µL of arabinose (200mg/mL) to the LB agar medium (in the case of PGLO)
  11. LB prepared with the bacterial solution after culture in 10. Apply 125μL to agar medium and incubate overnight at 37℃.

Plasmid extraction

  1. Centrifuge a 2mL tube containing the cultured bacterial solution (10000G, 3min, 4℃).
  2. Discard the supernatant so as not to break the pellet.
  3. Add 100pL of Solution I and confirm that the bacteria is suspended.
  4. Add 200pL of Solution II and mix slowly and gently. Make sure the solution is clear.
  5. Add 150pL of Solution II and mix slowly and gently. Confirm that it has become cloudy and solid matter has appeared.
  6. Let stand on ice for 5minutes.
  7. Centrifuge (10000rpm, 10min, 4℃).
  8. Take all of the supernatant and place in a new microtube to prevent precipitation.
  9. add 2μl of RNase (10mg/mL), then react it in heat block at 37℃ for 20minutes.
  10. Phenol: Add chloroform (1:1) in equal amounts to the supernatant and squeeze vigorously. Since it is an organic solvent, pipetting is performed several times when sucking to saturate the inside of the chip.
  11. Centrifuge (10000rpm, 5min, 4℃).
  12. Take all the supernatant without removing the intermediate layer and place in a new 1.5mL microtube.
  13. Add 200μL of chloroform to the microtube containing the supernatant, tap it, and push it.
  14. Centrifuge (10,000 x g, 4'℃, 1 min).
  15. After centrifugation, the supernatant is transferred to a new 1.5mL microtube.
  16. Add 3M sodium acetate to the microtube containing the supernatant so that the sucked supernatant: 3M sodium acetate becomes 10:1in ratio.
  17. Add 100% ethanol to a final concentration of 70% and squeeze well.
  18. centrifuge (10000rpm, 20min, 4℃).
  19. Discard the supernatant and Add 400μL of 70% ethanol. (Rinse)
  20. centrifuge (1000rpm, 20min, 4℃)
  21. Discard the supernatant, turn the microtube upside down on a Kim wipe, and dry it (5to10 min). The beret is easier to peel off, so never peel it off.
  22. Add 30pL of D2W to a dried microtube and dissolve the pellets by pipetting.
  23. Store frozen.

ligation

  1. Set the heat block to 16℃. (The temperature changes depending on the ligase used)
  2. Add 5μL of D2W.
  3. Add 5μL of ligation mix.
  4. Add 1μL of restriction enzyme-treated plasmid vector.
  5. Add 2μL of post-PCR insert to microtube.
  6. Set in heat block and let it stand in for 1 hour or more.

Gel purification

  1. dissolve 6×GR Mix Green Loading Buffer and Sample, let stand on ice for 5minutes.
  2. While waiting, set the heat block to 55℃.
  3. Apply the whole amount and electrophorese for about 20to30 min.
  4. After taking a picture, cut out the gel containing the target DNA.
  5. Place the cut-out gel in a new microtube.
  6. Add 500μL of FADF to the microtube and mix it using vortex(device).
  7. 10 to 15min until the gel dissolves. Incubate at 55℃.
  8. Confirm that the gel has melted.
  9. Cool for about 10minutes till room temperature.
  10. Attach the FADF column to the collection tube and centrifuge the entire volume.
  11. TIBITAN for 30seconds. Put 9μL of Sample into the column.
  12. Add the filtrate to the waste liquid.
  13. Add 750μL of Wash buffer.
  14. centrifuge for 30 sec with TIBITAN.
  15. Centrifuge the filtrate to the waste liquid.
  16. Centrifuge for 3min with TIBITAN.
  17. using a new 1.5mL tube, install the Elution Buffer (D2W Also possible) 40μL, apply to the center of the column.
  18. 2min left to settle.
  19. Centrifuge for 2min with TIBITAN.
  20. Make sure that the microtube contains the solution.
  21. Store frozen.