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Team:TU Darmstadt/Protocols/Electrocompetent cells
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Revision as of 14:59, 9 September 2012
Contents |
Electrocompetent cells
The transformation of E. coli with plasmid DNA via electroporation according to Dower et al. requires electrocompetent cells.
Materials
Equipment
- -80°C freezer
- Incubation shaker
- Centrifuge (cooling cababilities required!)
- OD meter
- Ice water bath
Chemicals & consumables
- Ice and/or liquid nitrogen
- Falcon tubes
- dYT Medium (50 ml p.c.)
- ice cold ddH2O (95+ ml p.c.)
Procedure
- Cultivate the cells in 50 ml dYT with a starting OD600 of 0.1 at 37°C and 180 U/min
- At an OD600 of 0.5 transfer the cells to a sterile Falcon tube
- harvesting of the cells is performed with centrifugation at 4000 U/min 4°C
- Wash the cells with an decreasing volume of 50/30/15 ml (discard the supernatant)
- use some of the supernatant in the last centrifugation step to resuspend the cells
Notes
- Important: Cooling must be maintained at all times.
