Electroporation

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This protocol is for transforming plasmid DNA into Escherichia coli cells.

Contents

Knight lab

Materials

For the following, you need one per DNA sample

Procedure

  1. Chill electroporation cuvettes, DNA samples and tubes on ice.
  2. Place LB-agar plates in 37°C incubator to warm.
  3. Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.
  4. If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled 0.6mL tubes.
  5. Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).
  6. Dial a P2 pipetman to either 1 or 2μL depending on the salt content of your DNA sample and . Use 2μL for samples that have been purified in some way.
  7. Dial a P200 pipetman to 50μL or whatever volume of electrocompetent cells you want to use. Usually 20-50μL.
  8. Dial a P1000 pipetman to 950μL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.
  9. Pipet 1-2μL of DNA sample and add to electrocompetent cells. Swirl tip around gently in cells to mix DNA and cells. Do not pipet up and down.
  10. Place cells back on ice to ensure they remain cold.
  11. Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold.
  12. Wipe off excess moisture from outside of cuvette.
  13. Place in chamber of electroporator.
  14. Slide the chamber in so that the cuvette sits snugly between electrodes.
  15. Pulse the cells with a shock by pressing button on electroporator.
  16. Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off.
  17. Transfer SOC-cell mixture to chilled eppendorf tube.
  18. Chill sample on ice for 2 mins to permit the cells to recover.
  19. Transfer eppendorf tube to 37°C incubator and shake to promoter aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
  20. Plate transformation onto prewarmed LB-agar plate supplemented with appropriate antibiotic. I generally plate 200μL but appropriate plating volume depends on efficiency of the transformation.
  21. Incubate plate overnight at 37°C.
  22. Leave remaining SOC-cell mixture on the benchtop overnight.
  23. If you don't have any transformants, plate the rest of the transformation in the morning.

Notes

If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampcillin takes a while to be pumped into cells at a high enough concentration to have an effect.

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