01/21/2013
Chemically Competent Cell Prep (BL21-DE3)
Haynes Lab, 2013
Prepare these solutions ahead of time and chill at 4°C overnight:
100 mM CaCl2, autoclaved
15% glycerol in sterile 100 mM CaCl2
- First autoclave the 100mM CaCl2
and then make 15% glycerol solution. (Do not autoclave the glycerol)
- Grow untransformed cells on plain LB (streak for single colonies).
- Inoculate 12.5 mL culture with colony from plate. Incubate with shaking at 37°C for 8 hours.
- Prepare two 250 mL of plane LB Broth and autoclave.
- Transfer 6.2 mL culture into 250 mL. Grow until absorbance of 0.6 A600 is reached. BL21 growth is slow, overnight incubation is recommended. (12hrs)
- Aliquot 125 mL culture to 4 sterile large 250mL polycarbonate spin bottles. Chill on ice for 15 min.
- Centrifuge cells at 4000 rpm for 10 min ate 4°C. (Dr. Wang's lab refrigerator centrifuge)
- Resuspend cells in sterile, ice-cold 100 mM CaCl2 by gentle pipetting (do not vortex!). Use 37.5 mL per 125mL culture (75 mL per 250 cell culture). Incubate on ice for 15 min.
- Centrifuge cells at 4000 rpm for 10 min at 4°C.
- Resuspend in sterile, ice-cold 100 mM CaCl2/15% glycerol (do not vortex!). Use 20 mL per 250 mL culture.
- The cell pellets are very sticky. We need to pipette up and down for a long time to get them completely resuspended.
- Leave on ice in a cold room/ fridge for 21 hours.
- Freeze 200μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C.
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PcTF Subcloning in E. coli
