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Bacterial transformation (heat shock)

Bacterial techniques

Standard heat-shock transformation of chemically competent E. coli. Use ~50 µL cells per reaction.

You'll need

• Chemically competent cells (DH5α, NEB 5α, Stbl3, Top10, etc.) — see making competent cells
• Plasmid DNA (1–10 ng for miniprepped, 1–5 µL for ligation/Gibson reactions)
• Pre-warmed SOC (or LB) at 37 °C
• Pre-warmed selective LB+antibiotic plates at 37 °C
• Heat block or water bath at 42 °C

Procedure

1. Thaw competent cells on ice (~10 min). Pre-chill 1.5 mL tubes on ice.
2. Aliquot 50 µL competent cells into each pre-chilled tube.
3. Add 1–5 µL DNA (do not exceed 5% of cell volume). Flick to mix; do not vortex.
4. Incubate on ice 30 min. (Quick mode: 5–10 min works for high-efficiency cells with miniprepped plasmid.)
5. Heat shock 42 °C, 30 s (NEB) or 45 s (DH5α from kit). Time precisely.
6. Return to ice 2 min.
7. Add 950 µL pre-warmed SOC.
8. Recover at 37 °C, 250 rpm, 1 h (60 min for kanamycin/spec/cm; 30 min for amp/carb).
9. Plate 50–200 µL on selective plates. For low-efficiency reactions, pellet cells (1 min, 5,000 × g), resuspend in 100 µL, plate all of it.
10. Incubate inverted at 37 °C, 14–18 h.

Controls (run every transformation)

• Negative: 50 µL cells + no DNA → expect zero colonies on selection.
• Positive: 50 µL cells + 10 pg pUC19 (or similar) → expect a lawn or hundreds of colonies; confirms competence.

Strain quick reference