Haynes:ChemComp cells

Chemically Competent Cell Prep

Haynes Lab, 2012

Prepare these solutions ahead of time and chill at 4°C overnight:
100 mM CaCl2, autoclaved
15% glycerol in sterile 100 mM CaCl2

1. Grow untransformed cells on plain LB (streak for single colonies).
2. Inoculate 25 mL culture with colony from plate. Incubate with shaking at 4°C for 4 hours.
3. Transfer 25 mL culture into 1 L. Grow until absorbance of 0.6 A600 is reached.
4. Aliquot 250 mL culture to 4 sterile large cone-bottom spin bottles (or several sterile 50 mL conicals). Chill on ice for 15 min.
5. Centrifuge cells at 4000 rpm for 10 min.
6. Resuspend cells in sterile, ice-cold 100 mM CaCl2 by gentle pipetting (do not vortex!). Use 75 mL per 250 mL culture. Incubate on ice for 15 min.
7. Centrifuge cells at 4000 rpm for 10 min.
8. Resuspend in 20 mL sterile, ice-cold 100 mM CaCl2/15% glycerol (do not vortex!).
9. Optional: Leave on ice in a cold room/ fridge 4 to 21 hours.
10. Freeze 100 – 500 μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C.