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Making chemically competent cells (Inoue method)

Bacterial techniques

Reproducibly yields ~10⁸–10⁹ CFU/µg pUC19 — sufficient for routine cloning. Plan for ~6 h hands-on across 2 days.

Solutions

TB (transformation buffer)

• 10 mM PIPES — 1.51 g
• 15 mM CaCl₂·2H₂O — 1.10 g
• 250 mM KCl — 9.32 g
• Dissolve in 450 mL H₂O. Adjust pH to 6.7 with KOH.
• Add 55 mM MnCl₂·4H₂O (5.44 g) AFTER pH is set.
• Bring to 500 mL. Filter-sterilise (0.22 µm). Store at 4 °C.

Procedure

1. Day 1: Streak DH5α (or your preferred strain) on an antibiotic-free LB plate. Incubate overnight at 37 °C.
2. Day 2 morning: Pick a single colony into 25 mL LB in a 250 mL flask. Shake at 37 °C until OD₆₀₀ reaches 0.3–0.5 (~3–4 h).
3. Inoculate 250 mL SOB (no Mg) in a 2 L flask with the entire starter. Shake at 18 °C until OD₆₀₀ = 0.55 (overnight, ~16–18 h).
4. Day 3: Chill flask on ice 10 min. Transfer to four pre-chilled 50 mL conical tubes.
5. Pellet at 2,500 × g, 4 °C, 10 min. Aspirate.
6. Resuspend each pellet gently in 20 mL ice-cold TB. Pool to two tubes (40 mL each).
7. Pellet again, 2,500 × g, 4 °C, 10 min. Aspirate.
8. Resuspend total pellet in 20 mL ice-cold TB. Add DMSO to 7% (1.4 mL DMSO into 20 mL).
9. Mix gently, incubate on ice 10 min.
10. Aliquot 50–100 µL into pre-chilled sterile 1.5 mL tubes on dry ice or in a −80 °C cooler block.
11. Store at −80 °C.

Notes

• All steps after the cold growth must be on ice; warming kills competence.
• Plasticware should be pre-chilled; centrifuge rotor pre-cooled.
• Always include a transformation control with a known-good plasmid (e.g., 10 pg pUC19) to track lot-to-lot quality.
• Don't refreeze a thawed aliquot. Discard.
• Quick alternative: TSS method for OK-but-not-great competent cells in 30 min — search Chung & Miller 1988.