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Team:Calgary/Notebook/Protocols/MakingChemicallyCompetentEcoliCells
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<li>Inoculate 5-10 mL LB at 37°C while shaking</li> | <li>Inoculate 5-10 mL LB at 37°C while shaking</li> | ||
| - | <li>Subculture 1 mL of bacteria solution into 50 mL LB broth at | + | <li>Subculture 1 mL of bacteria solution into 50 mL LB broth at 37°C; while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours) </li> |
<li>Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes </li> | <li>Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes </li> | ||
<li>Re-suspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li> | <li>Re-suspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li> | ||
Latest revision as of 00:13, 28 September 2013
Competent E. coli Cells
Making Chemically Competent E. coli Cells
Reagents and Materials
- Top10 E. coli cells
- LB Broth
- 50mM CaCl2 solution (store at 4°C)
Protocol
- Inoculate 5-10 mL LB at 37°C while shaking
- Subculture 1 mL of bacteria solution into 50 mL LB broth at 37°C; while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours)
- Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes
- Re-suspend pellet in 12.5 mL of cold CaCl2 (50 mM) and leave on ice for 10 minutes
- Centrifuge at 10 000 rpm at 4°C for 2 minutes and re-suspend in 2 mL of cold CaCl2 (50 mM, 15% glycerol solution)
- Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80°C
