Team:TU Dresden/Notebook/Protocols/Electroporation


Electroporation

  1. Set up an electroporator at 1,350 V.
  2. Add 1 μL of each plasmid to a different tube of electrocompetent cells.
  3. Add the whole mix into a precooled electroporation cuvette, carefully knock the cuvette on the table to remove air bubbles and dry the metallic sides of the cuvette.
  4. Place the cuvette into the holder of the electroporator, insert it and push 2 times the "pulse" button.
  5. Take the cuvette out of the electroporator and add 1 mL of pre-heated LB medium without antibiotics into the cuvette, mix carefully by slowly pipetting up and down, and transfer the solution back into an Eppendorf tube.
  6. Incubate the 37 °C (or at other temperatures depending on the sensitivity of the plasmid) for 45-60 minutes.
  7. Streak out 50 μL of electroporated cells onto an LB plate containing the specific antibiotic of the plasmid.
  8. Centrifuge the Eppendorf tube for 1 minute at 7,000 g and remove roughly 900 mL of the supernatant.
  9. Resuspend the cell pellet in the rest of the supernatant and streak it on a new LB plate containing the specific antibiotic.
  10. Incubate overnight at the specific temperature of the E. coli.