Haynes:ChemComp cells

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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 agar (streak for single colonies).
2. Inoculate 25 mL culture with colony from plate. Incubate with shaking at 37°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 sterile, ice-cold 100 mM CaCl₂/15% glycerol (do not vortex!). Use 20 mL per 250 mL culture.
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.