IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells

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Detailed below is the procedure to make CaCl2-competent stock cells. This is adapted from The Department of Biology at Indiana University.


  • 5 mL LB Media
  • 200 mL LB Media
  • Ice
  • Four 50 mL sterile centrifuge (conical) tubes
  • Centrifuge
  • 0.1 M CaCl2
  • 0.1 M CaCl2 / 15% glycerol


Day 1: Plate cells and grow overnight.

Day 2: Entire procedure should take approximately one hour.

  • Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).
  • Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of A600=0.6 is achieved; put on ice and keep chilled at 0ºC for 15 min.
  • Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.
  • Resuspend cells with 15 mL sterile, ice-cold 0.1M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave on ice 15 min. Spin again at 4000 rpm for 10 min.
  • Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).
*Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).

  • Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!



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