Ellis:Back Door/Protocols/Competent Cells

From OpenWetWare
Jump to navigationJump to search

Making Electrocompetent E. coli Cells

In order to maintain the efficiency of the cells, it is vital that once the mid-exponential culture is chilled, the cells remain at low temperature.

Day One

  1. Inoculate a 5 ml LB culture with a single colony of the E. coli strain and incubate O/N at 37 °C.
  2. Incubate a conical flask containing 500 ml of LB at 37 °C O/N.
  3. Store 500 ml sterile H2O in the fridge O/N

Day Two

  1. Use the O/N culture to inoculate the 37 °C LB broth 1:100 and incubate shaking at 37 °C.
  2. Get plenty of ice and pre-chill a sterile 20% (v/v) glycerol stock and the sterile H2O.
  3. Label microtubes and store in the -80 °C freezer.
  4. Pre-chill a rotor to 4 °C.
  5. When the OD600 of the culture reaches ~0.5, transfer to 50 ml Falcon tubes (ensure that there is no more than 40 ml/tube) and chill on ice for 30 minutes.
  6. Centrifuge the tubes in the rotor pre-chilled to 4 °C at 4000 rpm for 15 minutes.
  7. Discard the supernatant and, on ice, re-suspend the cells in the equivalent volume of pre-chilled water.
  8. Centrifuge as before.
  9. Discard the supernatant, on ice re-suspend cells in pre-chilled 20% glycerol (volume is not important but ideally just enough to re-suspend the cells e.g. 2ml/tube) and pool all of the cells into one of the 50 ml Falcon tubes.
  10. Centrifuge as before.
  11. Discard the supernatant and, on ice, re-suspend the cells in ~3 ml pre-chilled 20% glycerol.
  12. Transfer the cells into the pre-chilled microtubes in 50 μl aliquots and store immediately at -80 °C.