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.
- 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.
- Grow at 37 0C
overnight in shaking incubator.
- Add 1 mL of
overnight culture to 500 mL fresh LB in a 2L culture flask.
- Grow at 37 0C
for 2.5 – 3.5 hours in shaking incubator. (Check for log phase growth, OD600
= 0.375-0.5).
- Prepare
centrifuge during this time. Chill rotor.
- Transfer culture
to two centrifuge bottles. Keep on ice from this point forward.
- Centrifuge cold
5000-6000 rpm (JA-10) for 5 min.
- Pour off super
and resuspend cells in 50 mL cold 0.1 M CaCl2.
- 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.
- Leave to sit in
wet ice for 20 min.
- Centrifuge cold
5000-6000 rpm for 5 min.
- Pour off super,
add 4.5 mL 0.1 M CaCl2 and 6 mL 40% glycerol, resuspend cells
carefully.
- Transfer to 50
mL conical tube after resuspending.
- Pipette aliquots
(225 uL or so) into microtubes and quick freeze on dry ice, then store at
-80 0C.