Ellis:Back Door/Protocols/Competent Cells: Difference between revisions

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=Making Electrocompetent E. coli Cells=
=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==
<ol>
<ol>
<li>Get required amount of electrocompetent cells from the -80°C freezer and let them thaw slowly on ice. Usually 1 tube (50μL / tube) per transformation.</li>
<li>Inoculate a 5 ml LB culture with a single colony of the E. coli strain and incubate O/N at 37 °C.</li>
<li>Take the appropriate amount of [http://www.bio-rad.com/webroot/web/images/lsr/products/gene_transfer_rnai/product_detail/global/lsr_gp_cuvettes.jpg electroporation cuvettes] from the -20°C freezer (1 per transformation) and label them accordingly</li>
<li>Incubate a conical flask containing 500 ml of LB at 37 °C O/N.</li>
<li>Place 1-2μL of the DNA you wish to transform against the side of the cuvette, between the electrodes.</li>
<li>Store 500 ml sterile H2O in the fridge O/N</li>
<li>Add your competent cells to each transformation by targeting the drop of DNA previously deposited.</li>
</ol>
<li>Keeping the cuvettes on ice, add 50μL of the competent cells and mix by pipetting up and down trying to avoid creating air bubbles</li>
==Day Two==
<li></li>
<ol>
<li></li>
<li>Use the O/N culture to inoculate the 37 °C LB broth 1:100 and incubate shaking at 37 °C.</li>
<li>Get plenty of ice and pre-chill a sterile 20% (v/v) glycerol stock and the sterile H2O.</li>
<li>Label microtubes and store in the -80 °C freezer.</li>
<li>Pre-chill a rotor to 4 °C.</li>
<li>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.</li>
<li>Centrifuge the tubes in the rotor pre-chilled to 4 °C at 4000 rpm for 15 minutes.</li>
<li>Discard the supernatant and, on ice, re-suspend the cells in the equivalent volume of pre-chilled water.</li>
<li>Centrifuge as before.</li>
<li>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.</li>
<li>Centrifuge as before.</li>
<li>Discard the supernatant and, on ice, re-suspend the cells in ~3 ml pre-chilled 20% glycerol.</li>
<li>Transfer the cells into the pre-chilled microtubes in 50 μl aliquots and store immediately at -80 °C.</li>
</ol>
</ol>

Latest revision as of 03:27, 14 June 2011

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.
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