Ellis:Back Door/Protocols/Competent Cells

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(New page: =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....)
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=Making Electrocompetent E. coli Cells=
=Making Electrocompetent E. coli Cells=
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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.
 
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==Day One==
 
<ol>
<ol>
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<li>Inoculate a 5 ml LB culture with a single colony of the E. coli strain and incubate O/N at 37 °C.</li>
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<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>
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<li>Incubate a conical flask containing 500 ml of LB at 37 °C O/N.</li>
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<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>
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<li>Store 500 ml sterile H2O in the fridge O/N</li>
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<li>Place 1-2μL of the DNA you wish to transform against the side of the cuvette, between the electrodes.</li>
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</ol>
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<li>Add your competent cells to each transformation by targeting the drop of DNA previously deposited.</li>
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==Day Two==
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<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>
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<ol>
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<li></li>
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<li>Use the O/N culture to inoculate the 37 °C LB broth 1:100 and incubate shaking at 37 °C.</li>
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<li></li>
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<li>Get plenty of ice and pre-chill a sterile 20% (v/v) glycerol stock and the sterile H2O.</li>
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<li>Label microtubes and store in the -80 °C freezer.</li>
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<li>Pre-chill a rotor to 4 °C.</li>
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<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>
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<li>Centrifuge the tubes in the rotor pre-chilled to 4 °C at 4000 rpm for 15 minutes.</li>
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<li>Discard the supernatant and, on ice, re-suspend the cells in the equivalent volume of pre-chilled water.</li>
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<li>Centrifuge as before.</li>
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<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>
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<li>Centrifuge as before.</li>
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<li>Discard the supernatant and, on ice, re-suspend the cells in ~3 ml pre-chilled 20% glycerol.</li>
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<li>Transfer the cells into the pre-chilled microtubes in 50 μl aliquots and store immediately at -80 °C.</li>
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</ol>
</ol>

Revision as of 13:20, 13 June 2011

Making Electrocompetent E. coli Cells

  1. 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.
  2. Take the appropriate amount of electroporation cuvettes from the -20°C freezer (1 per transformation) and label them accordingly
  3. Place 1-2μL of the DNA you wish to transform against the side of the cuvette, between the electrodes.
  4. Add your competent cells to each transformation by targeting the drop of DNA previously deposited.
  5. Keeping the cuvettes on ice, add 50μL of the competent cells and mix by pipetting up and down trying to avoid creating air bubbles
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