CaCL2 Competent Cell Stock Protocol

Day 1

Plate cells and grow overnight

Day 2

Pick colony and grown in 5mL media o/n (Ab+)

Day 3

1. Add 1ml of o/n culture to 50ml media(ab+)
2. Grow cells until have O.D. 0.5-1.0
3. Spin cels at 6K rpm in sterile bottles
4. Resuspend with 25mL sterile, ice-cold 0.1M CaCl2 (DO NOT VORTEX)
5. Leave on ice 15 min
6. Spin again at 6K rpm (sterile bottles!)
7. Resuspend in 3.3ml (1/15volume) in sterile, ice-cold 0.1M CaCl2-15% glycerol (Do not Vortex)
8. Leave on ice 4 to 20 hours (BL21 for 4hrs, DH5alpha and XL-1 blue for o/n)
9. Freeze in sterile eppendorf tubes in labeled aliquots.