Mike Barnkob:Protocols/CRISPR/Transforming bacteria
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Protocol to chemically transform bacteria with constructs.
- OneShot Stbl3 chemical competent E. coli, including S.O.C media and control plasmid, pUC19.
- LB plates with antibiotic.
- Liquid LB media with antibiotic.
- Turn on waterbath to 42°C
- Warm up S.O.C. media
- Besides experimental constructs, transform an uncut backbone and one cut backbone (positive control), with nothing ligated into it (negative control).
- 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.
- Add 10 to 100ng of DNA pr. vial.
- Mix gently by shaking the tube.
Note: Companys protocol advises against pipetting up and down.
- Incubate cells on ice for 30 minutes.
- Heat-shock vials for 45 seconds in the 42°C waterbath.
- Place vials on ice for 2 minutes.
- Add 250μL pre-warmed S.O.C. media to each vial.
- Cap vials tightly and place in shaking incubator at 37°C for 1 hour.
- 15 minutes before incubation of cells is over, place LB plates in 37°C incubator to warm up.
- 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:
- Invitrogen OneShot Stbl3 protocol.