Team:Calgary/Notebook/Protocols/BacterialTransformation

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<li>Thaw 100μL of competent cells (per transformation) on ice just before they are needed</li>
<li>Thaw 100μL of competent cells (per transformation) on ice just before they are needed</li>
<li>Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li>
<li>Add DNA (max 20μl) thawed cells and mix by flicking the side of the tube. Leave on ice for 30 minutes</li>
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<li>Heat shock 5 minutes at 37 degrees Celsius</li>
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<li>Heat shock 5 minutes at 37°C</li>
<li>Place on ice for 5 minutes</li>
<li>Place on ice for 5 minutes</li>
<li>Add 250μl SOC medium to each tube</li>
<li>Add 250μl 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°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 50 μL on antibiotic plates</li>
<li>Plate approximately 50 μL on antibiotic plates</li>
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<li>Grow overnight at 37 degrees Celsius</li>
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<li>Grow overnight at 37°C</li>
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Latest revision as of 00:12, 28 September 2013

Bacterial Transformation

Reagents and Materials

  • Chemically competent E. coli
  • DNA (e.g., ligation reaction)
  • SOC medium
  • LB agar plates + desired antibiotic

Protocol

  1. Thaw 100μL 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°C
  4. Place on ice for 5 minutes
  5. Add 250μl 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 antibiotic plates
  9. Grow overnight at 37°C

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