IGEM:Paris Bettencourt 2012/Protocols/Competent Cells With CaCl2

From OpenWetWare

< IGEM:Paris Bettencourt 2012 | Protocols
Revision as of 11:29, 30 June 2012 by Denis. K. Samuylov (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Protocol: CaCl2 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.

NOTE: Never vortex competent cells.
Resuspend by pipetting with large Pasteur pipettes.

The night before  :

  1. The night before, inoculate a 5 ml culture and grow overnight with selection.

The day of  :

  1. 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.
  2. 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.
  3. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
    NOTE: Keep the cells at 4 ºC from now on.
  4. Resuspend cells in 15 ml, ice-cold 100 mM CaCl2.
    • Leave on ice 4 hours to overnight.
  5. Spin down the cells at 4 ºC, 4000 rpm, 15 minutes.
  6. Resuspend cells in 4 ml, ice-cold 100 mM CaCl2 + 15% glycerol.
  7. Aliquot into pre-chilled Eppendorf tubes. Use immediately or store at -80ºC.
NOTE: Frozen cells are only good once.
Do not refreeze cells once thawed.

Solution Recipe

Material
Amount
Notes
LB with antibiotics
50 ml
-
100 mM CaCl2
15 ml
Ice cold
100 mM CaCl2 + 15% Glycerol
4 ml
Ice cold
Personal tools