Electroporation of E. coli

Bacterial techniques

~10× higher transformation efficiency than chemical competence (10⁹–10¹⁰ CFU/µg). Use for libraries, large constructs, or recombineering.

Making electrocompetent cells

Inoculate 5 mL LB with your strain (DH10B, MG1655, NEB 10-beta, etc.). Grow overnight at 37 °C.
Dilute 1:100 into 500 mL LB in a 2 L flask. Shake at 37 °C until OD₆₀₀ = 0.5–0.6 (~2.5–3 h).
Chill flask on ice 30 min. From here on, keep everything ice-cold.
Pellet cells at 4,000 × g, 4 °C, 10 min. Aspirate.
Wash 1: resuspend in 500 mL ice-cold sterile 10% glycerol. Pellet. Aspirate.
Wash 2: resuspend in 250 mL ice-cold 10% glycerol. Pellet. Aspirate.
Wash 3: resuspend in 50 mL ice-cold 10% glycerol. Transfer to a 50 mL conical. Pellet 4,000 × g, 10 min.
Resuspend final pellet in ~2 mL 10% glycerol (volume = ~1/250 of original culture).
Aliquot 40–50 µL into pre-chilled tubes on dry ice. Store at −80 °C.

Thaw 1 aliquot on ice.
Pre-chill electroporation cuvette (1 mm gap) on ice.
Add 1–2 µL DNA (must be salt-free — eluted in H₂O or dialysed; otherwise you'll arc). Mix gently.
Transfer cells + DNA to the cuvette. Tap to settle to the bottom; no bubbles.
Wipe outside dry. Pulse: 1.8 kV (1 mm cuvette), 25 µF, 200 Ω. Time constant should be ~5 ms — if it's lower or the machine arcs, your DNA has too much salt.
Immediately add 950 µL pre-warmed SOC and transfer to a new 1.5 mL tube.
Recover 37 °C, 250 rpm, 1 h.
Plate dilutions on selective LB+antibiotic plates. For libraries, plate the entire culture.

Salt is the enemy. After ligation/Gibson, drop-dialyse on a 0.025 µm filter for 15 min, or column-clean and elute in water before electroporation.
If you arc: use less DNA, dilute the DNA in water, or check cuvette is dry on the outside.
Use 2 mm cuvettes at 2.5 kV if you must use higher salt.
For recombineering (lambda Red), use freshly prepared cells from cultures shifted to 42 °C for 15 min before harvesting.