Adapted from "A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: Application for DNA fragment transfer between chromosomes and plasmid transformation"

Yields 107–1011 transformants/µg replicative plasmid or 103 transformants/µg integrative plasmid.

For n transformations into a bacterial strain:

  1. Culture ≈ (1.5×109 × n) cells in liquid medium, for n · 50 µL transformations.
    This is the E. coli quantity in ≈3 mL OD 5 saturated LB culture per 50 µL transformation.
  2. Wash cells twice in ≥⅔ volume 300 mM sucrose, centrifuging room-temp at 16,000×g.

    • For higher efficiencies or to prevent sparking, wash cells once more in 300 mM sucrose.

    • Keep the sucrose sterile; contaminants can grow in it.

  3. Resuspend cells in 1∕60 volume 300 mM sucrose, yielding 109–1010 cells per 50 µL transformation (2×107 cells/µL)

  4. Transfer to 1 mM gap cold electroporation cuvette. Tap the cuvette hard twice upon bench to force cells to bottom and release bubbles.
  5. Wipe cuvette electrodes, and immediately electroporate at 1.8 kV, looking for a time constant ≥3.
  6. Recovery: Within a few seconds, resuspend the cells in 1 mL rich medium, transfer to culture tube, and recover 37°C shaking for 1–2 hr.
    • Or plate without recovery, yielding a few logs lower efficiency.
  7. Plate on appropriate agar, and incubate at growth-permissive temperature.
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