Difference between revisions of "Team:Birkbeck/Chemical Transformation"

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<h1>Transformation of plasmids into electrocompetent cells</h1>
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<h1>Chemical Transformation</h1>
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<p><b>See previous protocol: <a href="https://2015.igem.org/Team:Birkbeck/Chemical_Competent_E._coli_Cell_Protocol">Chemical Competent <i>E. coli</i> Cell Protocol</a></b> for details on making competent <i>E. coli</i> culls.</p>
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<h2><b>Electroporation protocol.</b></h2>
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<h2><b>Chemical Transformation protocol.</b></h2>
 
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<li>1. Thaw out the 50 µL cell suspension aliquot on ice for ca. 30 minutes. Concurrently, cool electroporation tubes on ice.</li>
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<li>1. Thaw out the 50 µL cell suspension aliquot on ice.</li>
 
<li>2. Add 100pg-1µg of plasmid DNA to the cell suspension (approximately 1-5 µL of plasmid solution).</li>
 
<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>3. Mix plasmid and cells thoroughly and continue to incubate on ice for 5-10 minutes.</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>4. Transfer the mixture of cells and plasmids to an electroporation tube.</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>5. Electroporate cells; for E.coli, this is done at 2.5 kV, 200 Ohms, 25 uF.</li>
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<li>5. Incubate on ice for 5 minutes.</li>
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<li>6. Immediately add 450-950 uL of LB broth.</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>7. Incubate tubes at 37<sup>o</sup>C for 60-90 minutes in a statis incubator.</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>8. Plate 100 uL of cells onto agar plates with appropriate antibiotic resistance.</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>9. Incubate overnnight at 37<sup>o</sup>C in a static incubator.</li>
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Revision as of 17:18, 17 September 2015

Chemical Transformation

See previous protocol: Chemical Competent E. coli Cell Protocol for details on making competent E. coli culls.

Chemical Transformation protocol.

  • 1. Thaw out the 50 µL cell suspension aliquot on ice.
  • 2. Add 100pg-1µg of plasmid DNA to the cell suspension (approximately 1-5 µL of plasmid solution).
  • 3. Mix plasmid and cells thoroughly and incubate on ice for 30 minutes.
  • 4. Induce heat shock by incubating cells at 42oC for 30 seconds.
  • 5. Incubate on ice for 5 minutes.
  • 6. Add 450 µL of LB and incubate at 37oC for 1 hour.
  • 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.
  • 8. Incubate overnnight at 37oC in a static incubator.