Difference between revisions of "Team:Birkbeck/Chemical Competent E. coli Cell Protocol"

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<h1>Making chemically competent cells</h1>
 
<h1>Making chemically competent cells</h1>
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<p><b>Unfinished!</b></p>
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<li>1. Thaw out the 50 µL cell suspension aliquot on ice.</li>
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<li>1. Grow 5 mL overnight culture in LB broth.</li>
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<li>2. Add 100pg-1µg of plasmid DNA to the cell suspension (approximately 1-5 µL of plasmid solution).</li>
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<li>2. Sub-culture (1% inoculum) in 25 mL of LB.</li>
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<li>3. Mix plasmid and cells thoroughly and incubate on ice for 30 minutes.</li>
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<li>3. Grow until OD600 = 0.3-0.5 (DH5a, this is around 2 hours).</li>
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<li>4. Induce heat shock by incubating cells at 42<sup>o</sup>C for 30 seconds.</li>
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<li>4. Put on ice for 20 mins (can be longer, not an essential step).</li>
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<li>5. Incubate on ice for 5 minutes.</li>
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<li>5. Spin cells down at 4<sup>o</sup>C at 4,500 rpm for 20 mins.</li>
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<li>6. Add 450 µL of LB and incubate at 37<sup>o</sup>C for 1 hour.</li>
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<li>6. Resuspend in 2.5 mL of ice cold TSB.</li>
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<li>7. Plate out 50 µL of straight transformant cells & also carry out a 10-fold dilution (plate out 50µL). Note that the plate should contain the appropriate antibiotic at the MBC in order to select for transformants which contain the desired plasmid.</li>
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<li>7. Leave on ice for at least 10 mins.</li>
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<li>8. Incubate overnnight at 37<sup>o</sup>C in a static incubator.</li>
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<li>8. Snap freeze in 50 uL aliquots to -80<sup>o</sup>C or use immediately.</li>
 
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Revision as of 21:48, 17 September 2015

Making chemically competent cells

  • 1. Grow 5 mL overnight culture in LB broth.
  • 2. Sub-culture (1% inoculum) in 25 mL of LB.
  • 3. Grow until OD600 = 0.3-0.5 (DH5a, this is around 2 hours).
  • 4. Put on ice for 20 mins (can be longer, not an essential step).
  • 5. Spin cells down at 4oC at 4,500 rpm for 20 mins.
  • 6. Resuspend in 2.5 mL of ice cold TSB.
  • 7. Leave on ice for at least 10 mins.
  • 8. Snap freeze in 50 uL aliquots to -80oC or use immediately.