IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells
Detailed below is the procedure to make CaCl2-competent stock cells.
- 5 mL LB Media
- 200 mL LB Media
- Four 50 mL sterile centrifuge (conical) tubes
- 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 .6A600 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 .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.
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