Team:Calgary/Notebook/Protocols/Process13

From 2011.igem.org

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   <ol>
   <ol>
     <li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li>
     <li> Thaw 100 μL of competent cells (per transformation) on ice just before they are needed</li>
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     <li> Add DNA (max 20ul) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li>
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     <li> Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li>
     <li> Heat shock 5 minutes at 37 degrees Celsius</li>
     <li> Heat shock 5 minutes at 37 degrees Celsius</li>
     <li> Place on ice for 5 minutes </li>
     <li> Place on ice for 5 minutes </li>
     <li> Add 250ul SOC medium to each tube</li>
     <li> Add 250ul SOC medium to each tube</li>
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     <li> Incubate for 30 to 60 minutes with shaking at 37 degrees Celsius. (Note that for Kanamycin containing plasmids always use one hour)</li>
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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> Spin down to remove all supernatant except approximately 100 μL</li>
     <li> Spin down to remove all supernatant except approximately 100 μL</li>
     <li> Plate approximately 30 μL on each of two antibiotic plates </li>
     <li> Plate approximately 30 μL on each of two antibiotic plates </li>
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     <li> Grow overnight at 37 degrees Celsius </li>
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     <li> Grow overnight at 37&deg;C </li>
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   </ol>
   <p> For this protocol we used a couple of controls
   <p> For this protocol we used a couple of controls
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Latest revision as of 04:21, 29 September 2011


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 30 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







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