Mike Barnkob:Protocols/CRISPR/Transforming bacteria

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Transforming bacteria

Protocol to chemically transform bacteria with constructs.

Reagents

  • OneShot Stbl3 chemical competent E. coli, including S.O.C media and control plasmid, pUC19.
  • LB plates with antibiotic.
  • Liquid LB media with antibiotic.

Protocol

Preparation:

  1. Turn on waterbath to 42°C
  2. Warm up S.O.C. media
  3. Besides experimental constructs, transform an uncut backbone and one cut backbone (positive control), with nothing ligated into it (negative control).

Transformation:'

  1. Thaw 1 vial of competent E.coli pr. transformation + 1 extra for positive control (pUC19). Keep cells on ice at all times.
    Note: Be careful to only handle the tubes near the top, as to not heat up the cells.
  2. Add 10 to 100ng of DNA pr. vial.
  3. Mix gently by shaking the tube.
    Note: Companys protocol advises against pipetting up and down.
  4. Incubate cells on ice for 30 minutes.
  5. Heat-shock vials for 45 seconds in the 42°C waterbath.
  6. Place vials on ice for 2 minutes.
  7. Add 250μL pre-warmed S.O.C. media to each vial.
  8. Cap vials tightly and place in shaking incubator at 37°C for 1 hour.
  9. 15 minutes before incubation of cells is over, place LB plates in 37°C incubator to warm up.
  10. Spread 50μL of each transformant onto warmed LB plates and incubate at 37°C over-night.
    Note: Store the LB plates inverted and with film around to inhibit drying out of the plates.
    Note: Transformation mix can be saved at 4°C till the next day.

Calculate transformation efficiency:


References

  • Invitrogen OneShot Stbl3 protocol.