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From 2013.igem.org

COMPETENT CELL PROTOCOL (Mike Scott)

1. The night before making competent cells, streak out desired strain on LB plate that is 10mM MgCl2 or MgSO4
2. Grow 3~5 fresh colonies of desired strain for 2 hours in 10 ml of TYM broth in 250 ml flask, 200rpm, 37℃ (aeration is very important)
3. Transfer cells to 200 ml fresh TYM broth in 1L flask. Shake for a further 2-3 hrs until Klett=40. (OD550 =0.5)
4. Spin cells down in four 50 ml polypropylene tubes, in SS34 rotor at 3K rpm for 10-12 min, just enough to pellet the cells.
5. Drain cells and re-suspend in 30-40 ml per 100 ml starting culture of TfbI. Leave on ice. For DH1 the optimal time is 5-10 min; good results are obtained for MC1061 when it is left 5 min on ice.For HB101 the time is 90-120 min. For DH5α the time is 90-120 min. For BL21 the time is 25-30 min. Other strains should be assayed for optimal time.
6. Spin cells down, 3K rpm for 8 min at 4℃.
7. Resuspend in 4 ml per 100ml starting culture of TfbII on ice. Be gentle. Aliquot 0.1 ml into eppendorf tubes for DH5α,on ice. Aliquot 0.05 ml into eppendorf tubes for BL21,on ice,store at-80℃.