Preparing CaCl2 Competent E. coli Cells

2004-03-12 (from Chris Udell)

 

Required: 500 mL LB (plus a little), autoclaved 2 L culture flask, autoclaved large (250 or 500 mL) centrifuge bottles, sterile 0.1 M CaCl2, sterile 40% glycerol solution.

 

  1. Innoculate 5 mL LB broth with E. coli culture. Ideally, streak a plate and then pick one colony. Alternatively, use a micropipette tip to scrape some cells from the top of a glycerol stock.
  2. Grow at 37 0C overnight in shaking incubator.
  3. Add 1 mL of overnight culture to 500 mL fresh LB in a 2L culture flask.
  4. Grow at 37 0C for 2.5 3.5 hours in shaking incubator. (Check for log phase growth, OD600 = 0.375-0.5).
    1. Prepare centrifuge during this time. Chill rotor.
  5. Transfer culture to two centrifuge bottles. Keep on ice from this point forward.
  6. Centrifuge cold 5000-6000 rpm (JA-10) for 5 min.
  7. Pour off super and resuspend cells in 50 mL cold 0.1 M CaCl2.
    1. Add small volume of CaCl2 and gently pipette up and down, mix cells, etc. until most of the pellet is resuspended, then add the rest of the CaCl2 and shake a bit to mix.
  8. Leave to sit in wet ice for 20 min.
  9. Centrifuge cold 5000-6000 rpm for 5 min.
  10. Pour off super, add 4.5 mL 0.1 M CaCl2 and 6 mL 40% glycerol, resuspend cells carefully.
    1. Transfer to 50 mL conical tube after resuspending.
  11. Pipette aliquots (225 uL or so) into microtubes and quick freeze on dry ice, then store at -80 0C.