Team:Calgary/Notebook/Protocols/MakingChemicallyCompetentEcoliCells

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<li>Top10 <i>E. coli</i> cells</li>
<li>Top10 <i>E. coli</i> cells</li>
<li>LB Broth</li>
<li>LB Broth</li>
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<li>50mM CaCl<sub>2</sub> solution (store at 4 degrees Celsius)</li>
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<li>50mM CaCl<sub>2</sub> solution (store at 4°C)</li>
</ul>
</ul>
<h2>Protocol</h2>
<h2>Protocol</h2>
<ol>
<ol>
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<li>Inoculate 5-10 mL LB at 37 degrees Celsius while shaking</li>
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<li>Inoculate 5-10 mL LB at 37°C while shaking</li>
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<li>Subculture 1 mL of bacteria solution into 50 mL LB broth at 37 degrees Celsius; while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours) </li>
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<li>Subculture 1 mL of bacteria solution into 50 mL LB broth at 37°C; while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours) </li>
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<li>Centrifuge the subculture at 10 000 rpm at 4 degrees Celsius for 2 minutes </li>
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<li>Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes </li>
<li>Re-suspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li>
<li>Re-suspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li>
-
<li>Centrifuge at 10 000 rpm at 4 degrees Celsius for 2 minutes and re-suspend in 2 mL of cold CaCl<sub>2</sub> (50 mM, 15% glycerol solution) </li>
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<li>Centrifuge at 10 000 rpm at 4°C for 2 minutes and re-suspend in 2 mL of cold CaCl<sub>2</sub> (50 mM, 15% glycerol solution) </li>
-
<li>Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80 degrees Celsius</li>
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<li>Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80°C</li>
</ol>
</ol>
</section>
</section>
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Latest revision as of 00:13, 28 September 2013

Making Chemically Competent E. coli Cells

Reagents and Materials

  • Top10 E. coli cells
  • LB Broth
  • 50mM CaCl2 solution (store at 4°C)

Protocol

  1. Inoculate 5-10 mL LB at 37°C while shaking
  2. Subculture 1 mL of bacteria solution into 50 mL LB broth at 37°C; while shaking until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours)
  3. Centrifuge the subculture at 10 000 rpm at 4°C for 2 minutes
  4. Re-suspend pellet in 12.5 mL of cold CaCl2 (50 mM) and leave on ice for 10 minutes
  5. Centrifuge at 10 000 rpm at 4°C for 2 minutes and re-suspend in 2 mL of cold CaCl2 (50 mM, 15% glycerol solution)
  6. Leave on ice for at least 30 minutes and then aliquot 200 μL and freeze at -80°C

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