IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells

Overview

Detailed below is the procedure to make CaCl₂-competent stock cells.

Materials

• 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

Procedure

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.

Notes

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