IGEM:IMPERIAL/2009/Transforming Competent Cells

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Aims

To transform our electrocompetent E. coli with plasmids containing selected DNA/Parts/BioBricks.

Equipment

  • Desktop centrifuge
  • Electroporation machine
  • Electrocuvettes
  • Eppendorf tubes
  • P1000 and P200 Gilsons and tips

Reagents

  • XL-1 Blue competent cells (stored in the freezer)
  • LB
  • Plates with required antibiotic

Protocol

  • 1ul DNA into competent cell suspension. Pipette up and down.
  • Take 50ul – put into electro-cuvette. Tap to ensure even distribution.
  • Electroporate – make sure the machine is set to ‘bacteria’ on the mV setting. You should get a reading between 3.00 and 4.00 on the m/s read-out.
  • Add 450ul LB to cuvette. Pipette up and down.
  • Take 450ul and put into a new eppendorf tube. Put in the shaking incubator for 30mins.
  • Centrifuge for 1minute.
  • Discard supernatant.
  • Plate out cells.
  • Incubate overnight at 37°C