IGEM:Paris Bettencourt 2012/Protocols/Competent Cells With CaCl2
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Protocol: [math]\displaystyle{ CaCl_{2} }[/math] Competent Cells
This protocol makes 4 ml of competent cells, and can be easily scaled up to make more. The cells are typically stored in 110 ul aliquots, so this will make about 35 tubes. A typical transformation uses 20 ul of cells.
The night before :
- The night before, inoculate a 5 ml culture and grow overnight with selection.
The day of :
- Dilute cells ~ 1:200 into selective media.
- For this example add 250 ul to 50 ml of selective media.
- NOTE: The protocol is easily scaled to increase the number of cells.
- Grow the cells to an Optical Density (OD, 600 nm) of 0.6 – 0.7.
- Use a large flask, 500 ml, for good aeration.
- Use a baffled flask for fastest growth.
- This takes about 3 hours depending on the cells.
- Medium-heavy cloudiness by eye is fine.
- Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
NOTE: Keep the cells at 4 ºC from now on. - Resuspend cells in 15 ml, ice-cold 100 mM CaCl2.
- Leave on ice 4 hours to overnight.
- Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
- Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
- Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.