Difference between revisions of "Team:Freiburg/Protocols/Transformation"

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Revision as of 07:03, 20 November 2015

""

Transformation

Introducing plasmid DNA into bacteria

material: chemical competent cells, heatblock (42°C), shaker (37°) LB plates (with antibiotics), LB-Medium (without entibiotics), ice
duration: 2 h

Trafo

  1. add 1 µL plasmid DNA to 25 µL chemical competent cells (thaw on ice and handle gently)
  2. incubate 10 min on ice
  3. heat shock 45 sec 42°C
  4. incubate 2 min on ice
  5. add 500 µL LB medium (without antibiotics)
  6. incubate 1 h, 37°C (for re-trafo 15 min is sufficient)
  7. centrifuge 4000 rpm, 1min (discard supernatant, resuspend in remaining medium)
  8. plate 15 µL on LB-plates with antibiotics
  9. incubate over night at 37°C

Gibson trafo

  1. add 5 µL Gibson mix to 25 µL chemical competent cells (thaw on ice and handle gently)
  2. heat shock 45 sec 42°C
  3. incubate 2 min on ice
  4. add 500 µL LB medium (without antibiotics)
  5. incubate 1 h, 37°C
  6. plate 300 µL on LB-plates with antibiotics
  7. incubate over night at 37°C