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Team:SDU-Denmark/Protocols/Competent-cells
From 2009.igem.org
Protocol for making cells (E. coli) competent
(Cells are kept on ice at all times!! If the temperature rises above ~5˚C they lose their competency)
- 2ml over-night cell culture is transferred to 200ml Luria Bertani (LB)-medium.
- Incubate at 37˚C on shaker until OD550=0.5-0.7
- Keep on ice for 15-30min and harvest by 4000 x g for 15 min at 4˚C.
- Remove all supernatant and resuspend carefully in 200ml ice cold dH2O. Harvest as in 3.
- Resuspend in 100ml ice cold dH2O. Harvest as in 3.
- Resuspend in 20ml ice cold 10% glycerol. Harvest as in 3.
- Resuspend in ice cold 10% glycerol until a total volume of 1ml. Cell concentration should be 1-3x1010 cells/ml.
- Cells are distributed in tubes with 40ul in each and kept at -80˚C.
