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Synthetic Biology

Genome Extraction

Material
Items (Volume)
Buffer GA (200ul)
Proteinase K solution (20ul)
Buffer GB (220ul)
Ethanol (220ul)
Buffer GD (500ul)
Rinse PW (600ul)
Elution Buffer TE (100ul)

Extraction steps:

  1. Absolute ethanol before use in buffer and rinse GD PW, adding volume refer to the label on the bottle.

  2. Take inoculum 1-5 ml, 10,000 rpm (~ 11,500 × g) was centrifuged 1 min, the supernatant net absorption as possible.

  3. The cell pellet is added to 200 μl buffer GA, shaking to complete the cell suspension.

  4. The tube is added to 20 μl Proteinase K solution, mixed evenly.

  5. Add 220 μl buffer GB, oscillation 15 sec, 70 ℃ and placed 10 min, strain the solution clear, brief centrifugation to remove the tube drops on the inner wall .

  6. Add 220 μl ethanol, mix thoroughly shaken 15 sec, flocculent precipitate may occur at this time, a brief centrifugation to remove the tube drops on the inner wall .

  7. Previous resulting solution and the flocculent precipitate are added into a adsorption column CB3(adsorption column into the collection tube), 12,000 rpm centrifugation 30 sec, discard the liquid, set the adsorption column CB3 into the collection tube.

  8. 500 μl buffer GD was added to CB3 adsorption column(check whether ethanol has been added before use), 12,000 rpm (~ 13,400 × g) centrifugation 30 sec, discard the liquid, set the adsorption column CB3 into the collection tube.

  9. 600 μl rinse PW was added to CB3 adsorption column(check whether ethanol has been added before use), 12,000 rpm(~ 13,400 × g) centrifugation 30 sec, discard the liquid, set the CB3 adsorption column into the collection tube.

  10. Repeat steps 8.

  11. Place the column back into the collection tube CB3, 12,000 rpm (~ 13,400 × g) centrifugation 2 min, discard the waste.Place the adsorption column CB3 at room temperature for several minutes to completely dry absorbent material.

  1. Put the CB3 adsorption column into a clean centrifuge tube and drop 50-200 μl elution buffer TE to the middle of the adsorbed film vacant. Put at room temperature 2-5 min, 12,000 rpm (~ 13,400 × g) centrifugation 2 min, collect the solution in a centrifuge tube.

  2. Detect the concentration of DNA.