Team:Calgary/Notebook/Protocols/transformation

From 2012.igem.org

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     <li> Incubate for 30 to 60 minutes with shaking at 37&deg;C. (Note that for Kanamycin containing plasmids always use one hour)</li>
     <li> Incubate for 30 to 60 minutes with shaking at 37&deg;C. (Note that for Kanamycin containing plasmids always use one hour)</li>
     <li> Spin down to remove all supernatant except approximately 100 μL</li>
     <li> Spin down to remove all supernatant except approximately 100 μL</li>
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     <li> Plate approximately 30 μL on each of two antibiotic plates </li>
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     <li> Plate approximately 50 μL on each of two antibiotic plates </li>
     <li> <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Grow overnight</a> at 37&deg;C </li>
     <li> <a href="http://2012.igem.org/Team:Calgary/Notebook/Protocols/onculture">Grow overnight</a> at 37&deg;C </li>
   </ol>
   </ol>

Latest revision as of 03:29, 4 October 2012

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Bacterial Transformation

  1. Thaw 100 μL of competent cells (per transformation) on ice just before they are needed
  2. Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes
  3. Heat shock 5 minutes at 37 degrees Celsius
  4. Place on ice for 5 minutes
  5. Add 250ul SOC medium to each tube
  6. Incubate for 30 to 60 minutes with shaking at 37°C. (Note that for Kanamycin containing plasmids always use one hour)
  7. Spin down to remove all supernatant except approximately 100 μL
  8. Plate approximately 50 μL on each of two antibiotic plates
  9. Grow overnight at 37°C

For this protocol we used a couple of controls

  • Positive Control - pBluescript in TOP10 cells on ampicillin plates
  • Negative Control - TOP10 cells grown on ampcillin plates