IGEM:IMPERIAL/2006/Protocols/competent cells DH5a

Competent Cells (E.coli DH-5α)

1. Plate E.coli DH5 on an agar plate (no antibiotics)
2. Grow 2ml of E.coli DH-5α O/N in a 50ml Falcon tube.
3. Pour 400ml of BHI in each of two 2L conical flask.
4. Add 400μL of O/N culture into each flask using filter tips.
5. Incubate at 37[[:Category:{{{1}}}|{{{1}}}]] for about 4hours until the OD (600nm) reaches 0.7-1.0. Blank either with BHI or MiliQ. (Use sterile 5ml pipette!)
6. After incubation, place each flask in ice box and leave to chill for about 1hr
7. Transfer into 50ml Falcon tubes (8 of them: each tube contain 100ml worth of cell culture)
8. Spin for 5min at 5K at 4[[:Category:{{{1}}}|{{{1}}}]]. (programme 2)
9. Discard supernatant, resuspend in 5ml-ish MiliQ (in the cold cabinet) and vortex well to resuspend the pellet. Top it up to 50ml with MiliQ and spin as above. Carry out this step twice reducing the number of tubes by combining samples.
10. Wash with 10% glycerol (top shelf, cold cabinet), pooled into 2 tubes. Top up the tube with 10% glycerol and spin at 5K, 4[[:Category:{{{1}}}|{{{1}}}]] for 10min. [ programme 2 and then increase the time]
11. Resuspend pellet in 800μL (into each tube) 10% glycerol using filtered pipette.
12. Aliquot 50μL into eppendorf tubes on dry ice. Use new syringe with blue tips.
13. Test: positive control + negative control
    1. Positive: Use a plasmid. After zapping, add 1ml LB, don’t spin just plate about 50μL.
    2. Negative: Just the E.coli cells.