Team:BIOTEC Dresden/Protocols:Electroporation for normal samples



  • Escherichia coli DH5 alpha electrocompetent cells
  • Plasmid DNA
  • Ice
  • 2mm gap width Electroporation cuvette
  • Electroporater
  • 1.5ml microfuge tube
  • 1mL SOC (room temperature) for each reaction
  • LB-agar plate with corresponding antibiotic


  • Chill electroporation cuvettes, DNA samples and tubes on ice.
  • Place LB-agar plates in 37°C incubator to warm.
  • Once cuvettes are cold, remove electrocompetent cells from -80°C freezer and thaw on ice. Alternatively, freshly prepared electrocompetent cells may be used immediately.
  • If electrocompetent cells are not already in individual aliquots, then aliquot out into pre-chilled 0.6mL tubes.
  • Turn on electroporator and set voltage to either 1.25 kV (1mm cuvettes) or 2.5 kV (2mm cuvettes).
  • 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.
  • Dial a P200 pipetman to 50μL or whatever volume of electrocompetent cells you want to use. Usually 20-50μL.
  • Dial a P1000 pipetman to 950μL and pipet in SOC. Place pipetman on counter such that tip doesn't touch anything.
  • 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.
  • Place cells back on ice to ensure they remain cold.
  • Transfer cell-DNA mixture to cuvettes using P200 pipetman. Try not to handle cuvette base too much so that it stays cold.
  • Tap the cuvette on the counter gently so that cells are at the bottom and to remove any air bubbles.
  • Wipe off excess moisture from outside of cuvette.
  • Place in chamber of electroporator.
  • Slide the chamber in so that the cuvette sits snugly between electrodes.
  • Pulse the cells with a shock by pressing button on electroporator.
  • Remove cuvette from the chamber and immediately add SOC. This step should be done as quickly as possible to prevent cells from dying off.
  • Transfer SOC-cell mixture to chilled eppendorf tube.
  • Chill sample on ice for 2 mins to permit the cells to recover.
  • Transfer eppendorf tube to 37°C incubator and shake to promote aeration. Incubate for 1 hr to permit expression of antibiotic resistance gene.
  • 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.
  • Incubate plate overnight at 37°C.
  • Leave remaining SOC-cell mixture on the benchtop overnight.
  • If you don't have any transformants, plate the rest of the transformation in the morning.


adapted from

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