Team:HokkaidoU Japan/Notebook/overall protocols

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Transformation

  1. Add 1~2 ul of DNA to 50 ul of thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Add 600 ul of LB.
  4. Incubate the cells for 2 hours at 37C if added DNA plasmid have antibiotic resistance other than ampicillin.
  5. Prepare and Label two plastic plates with LB and appropriate antibiotic.
  6. Plate 300 ul of the culture onto first dish and spread.
  7. Add 900 ul of LB to 100 ul of the culture and plate 300 ul of it onto second dish and spread.
  8. Incubate the plates at 37C for 16~20 hours.

Mini-prep

We use mini-prep kit of Nippon genetics: FastGene Plasmid mini kit. This kit contains these reagents and wares: mP1, mP2, mP3, mP4, mP5, mP6, column and collection tube.

  1. Centrifuge 1~5 ml of culture at over 10,000 rpm for 2 min.
  2. Remove the supernatant.
  3. Add 200ul of mP1 then voltexing.
  4. Add 200ul of mP2 and invert the tube then leave for 2 min at room temperature.
  5. Add 200ul of mP3 then invert the tube.
  6. Centrifuge at 1,3000 rpm for 8 min.
  7. Centrifuge 1,3000 rpm for 1 min to load supernatant from column to collection tube.
  8. Remove filtrate and add 400 ul of mP4 then centrifuge 13,000 rpm for 1 min.
  9. Remove filtrate and add 600 ul of mP5 then centrifuge 13,000 rpm for 1 min.
  10. Remove filtrate and centrifuge 13,000 rpm for 2 min.
  11. Set column into 1.5 ml centrifuge tube.
  12. Add 50 ul of mP6.
  13. Centrifuge at 13,000 rpm for 2 min.



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