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.

References