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

(Created page with "{{BirkbeckNavigationBar}} <html> <div id="maincontainer"> <h1>Chemical Transformation</h1> <p><b>See previous protocol: <a href="https://2015.igem.org/Team:Birkbeck/Chemical_Co...")
 
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<h1>Chemical Transformation</h1>
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<h1>Making chemically competent cells</h1>
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<p><b>See previous protocol: <a href="https://2015.igem.org/Team:Birkbeck/Chemical_Competent_E._coli_Cell_Protocol"></a>Chemical Competent <i>E. coli</i> Cell Protocol</b> for details on making competent <i>E. coli</i> culls.</p>
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<p><b>Unfinished!</b></p>
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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.</li>
 
<li>1. Thaw out the 50 µL cell suspension aliquot on ice.</li>

Revision as of 18:16, 17 September 2015

Making chemically competent cells

Unfinished!

  • 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.