Preparing Calcium Chloride Competent Cells

CaCl2 Competent Cell Protocol

Purpose

This is a fast and fairly efficient way to remove the cell walls from E. coli so that they can be transformed with plasmids. This particular chemical competency method works very well on B strain (REL606) E. coli, but not very well on K12 types.

Materials

  • 2 ice buckets (one for icing cells/media, one for the dry ice/ethanol bath)
  • sterile 15mL and 50mL centrifuge tubes
  • sterile 1.5mL epis
  • pipettes and sterile tips
  • sterile flasks and tubes
  • labeled freezer box
  • laboratory ice
  • 70% ethanol
  • dry ice

Media

  • LB broth
  • 40mL of 0.1M CaCl2
    Mix 36mL of LB and 4mL of 1M CaCl2 in a 50mL centrifuge tube and put on ice.
  • 10mL of 0.1 M CaCl2/15% glycerol
    Mix 7.12mL of LB with 1.88mL of 80% glycerol and 1mL of 1M CaCl2 in a 15mL centrifuge tube and put on ice.

Equipment

  • Centrifuge with refrigeration capable of spinning 50mL tubes up to 4000rpm
  • Cuvette or plate reader to measure OD

Methods

Day 1

Inoculate 5mL of LB in an 18mm tube with a scraping of your bacteria from the freezer and incubate in the 37°C shaker overnight.

Day 2

Get a bucket of ice first thing. Make all media in centrifuge tubes so you can ice it while your cells are growing. Once you start it is important to keep everything as cold as possible.

  1. Transfer 100µL from your overnight culture into 50mL LB media in a 125mL flask and return to shaker for around 3 hours until an OD600 of 0.4 to 0.6 is achieved. Test the OD periodically by pipetting 300µL of your culture into a microtiter well and taking a static read at OD600 on the Versa Max.
  2. Transfer culture to 50mL centrifuge tube and put on ice for 15 minutes. Turn on the centrifuge, put in the 50mL tube buckets, and set it to 4°C.
  3. Spin cells at 4000 rpm for 10 min at 4°C and discard supernatant into a waste beaker.
  4. Re-suspend cells in 15mL sterile, ice-cold .1M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave on ice 15 min.
  5. Spin again at 4000 rpm for 10 min at 4°C. Discard supernatant into waste beaker.
  6. Re-suspend in 4mL sterile, ice-cold 0.1 M CaCl2/15% glycerol by gently pipetting (DO NOT VORTEX). Cells can be left on ice now for up to 4 hours to improve efficiency.
  7. Aliquot into 1.5mL epis with 50-100µL each and flash freeze in a dry ice/ethanol bath. Store cells in the -80°C freezer. Do not make your aliquots too large, as cells, once unfrozen, must be used immediately and should not be put back in the freezer.

Dry Ice/Ethanol Bath

To make a dry ice/ethanol bath, simply place several chunks of dry ice in a large, leak proof container with a lid (like a rubber ice bucket) and pour enough 70% ethanol on them until they are partially submerged. Use gloves to put the epis in the ice bath, as the ethanol around the dry ice will get extremely cold (cold enough to freeze the cells in the epis in under a minute).


Original protocol by: H. Lindsey Date: 11 Sept 12

Protocol Updated by: C. Eshelman Date: 13 Sept 12

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