Haynes:ChemComp cells: Difference between revisions
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15% glycerol in sterile 100 mM CaCl<sub>2</sub><br><br> | 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 4°C for 4 hours. | # Inoculate 25 mL culture with colony from plate. Incubate with shaking at 4°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. |
Revision as of 17:04, 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 4°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.