Haynes:ChemComp cells: Difference between revisions
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=Chemically Competent Cell Prep= | =Chemically Competent Cell Prep= | ||
Haynes Lab, 2012 | Haynes Lab, 2012 | ||
Prepare these solutions ahead of time and chill at 4°C overnight:<br> | Prepare these solutions ahead of time and chill at 4°C overnight:<br> | ||
100 mM | 100 mM CaCl<sub>2</sub>, autoclaved<br> | ||
15% glycerol in sterile 100 mM | 15% glycerol in sterile 100 mM CaCl<sub>2</sub><br><br> | ||
# Grow untransformed cells on plain LB (streak for single colonies). | # Grow untransformed cells on plain LB agar (streak for single colonies). | ||
# Inoculate 25 mL culture with colony from plate. Incubate with shaking at | # Inoculate 25 mL culture with colony from plate. Incubate with shaking at 37°C for 4 hours. | ||
# Transfer 25 mL culture into 1 L. Grow until absorbance of 0.6 A600 is reached. | # Transfer 25 mL culture into 1 L. Grow until absorbance of 0.6 A600 is reached. | ||
# Aliquot 250 mL culture to 4 sterile large cone-bottom spin bottles (or several sterile 50 mL conicals). Chill on ice for 15 min. | # Aliquot 250 mL culture to 4 sterile large cone-bottom spin bottles (or several sterile 50 mL conicals). Chill on ice for 15 min. | ||
# Centrifuge cells at 4000 rpm for 10 min. | # Centrifuge cells at 4000 rpm for 10 min. | ||
# Resuspend cells in sterile, ice-cold 100 mM | # Resuspend cells in sterile, ice-cold 100 mM CaCl<sub>2</sub> by gentle pipetting (do not vortex!). Use 75 mL per 250 mL culture. Incubate on ice for 15 min. | ||
# Centrifuge cells at 4000 rpm for 10 min. | # Centrifuge cells at 4000 rpm for 10 min. | ||
# Resuspend in | # Resuspend in sterile, ice-cold 100 mM CaCl<sub>2</sub>/15% glycerol (do not vortex!). Use 20 mL per 250 mL culture. | ||
# Optional: Leave on ice in a cold room/ fridge 4 to 21 hours. | # Optional: Leave on ice in a cold room/ fridge 4 to 21 hours. | ||
# Freeze 100 – 500 μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C. | # Freeze 100 – 500 μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C. | ||
Latest revision as of 17:05, 11 April 2013
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
- Grow untransformed cells on plain LB agar (streak for single colonies).
- Inoculate 25 mL culture with colony from plate. Incubate with shaking at 37°C for 4 hours.
- Transfer 25 mL culture into 1 L. Grow until absorbance of 0.6 A600 is reached.
- Aliquot 250 mL culture to 4 sterile large cone-bottom spin bottles (or several sterile 50 mL conicals). Chill on ice for 15 min.
- Centrifuge cells at 4000 rpm for 10 min.
- 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.
- Centrifuge cells at 4000 rpm for 10 min.
- Resuspend in sterile, ice-cold 100 mM CaCl2/15% glycerol (do not vortex!). Use 20 mL per 250 mL culture.
- Optional: Leave on ice in a cold room/ fridge 4 to 21 hours.
- Freeze 100 – 500 μL aliquots of cells in sterile microcentrifuge tubes. Store at -80°C.
