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Team:Calgary/Notebook/Protocols/compcells
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<li> Resuspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li> | <li> Resuspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li> | ||
<li> Centrifuge at 10 000 rpm at 4°C for 2 minutes and resuspend in 2 mL of cold CaCl<sub>2</sub> (50 mM, 15% glycerol solution) </li> | <li> Centrifuge at 10 000 rpm at 4°C for 2 minutes and resuspend in 2 mL of cold CaCl<sub>2</sub> (50 mM, 15% glycerol solution) </li> | ||
| - | <li> Leave on ice for at least 30 minutes and then aliquot 200 | + | <li> Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80°C </li> |
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Latest revision as of 03:37, 4 October 2012
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Making Chemically Competent E. coli Cells
This procedure was done using Top10 cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.
- Innoculate 5-10 mL LB at 37°C while shaking
- Subculture 1 mL of bacteria solution into 50 mL LB broth at 37° 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
- Resuspend 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 resuspend 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
