Before you start
- Chill all solutions, tubes, pipettes and the centrifuge in advance.
Reagents and materials
Totals are for 1 cultures (+10% extra). Per-100 mL culture (≈ 40 aliquots) amounts are in the middle column.
| Reagent | Per 100 mL culture (≈ 40 aliquots) | Total |
|---|---|---|
| LB (or SOB) medium | 100 mL | 100 mL |
| Ice-cold 0.1 M CaCl₂ | 50 mL | 50 mL |
| Ice-cold 0.1 M CaCl₂ + 15% glycerol | 2 mL | 2 mL |
| Pre-chilled microtubes | 40 each | 40 each |
Buffers and recipes
0.1 M CaCl₂ per 500 mL
| CaCl₂·2H₂O | 7.35 g |
| Ultrapure water | to 500 mL |
Filter-sterilise and chill on ice overnight.
Equipment
- Shaking incubator
- Spectrophotometer
- Refrigerated centrifuge with pre-chilled bottles
- Ice bucket
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Grow
Inoculate 100 mL medium 1:100 from an overnight culture and grow at 37 °C with shaking to OD₆₀₀ 0.3–0.4 (mid-log).
Chill
Chill the culture on ice for 20–30 min.
Pellet
Centrifuge at 4,000 × g for 10 min at 4 °C.
First CaCl₂ wash
Gently resuspend in 50 mL ice-cold 0.1 M CaCl₂ and keep on ice for 30 min.
Pellet again
Centrifuge at 4,000 × g for 10 min at 4 °C.
Final resuspension
Gently resuspend in 2 mL ice-cold 0.1 M CaCl₂ + 15% glycerol.
Aliquot and freeze
Dispense 50 µL into pre-chilled tubes and freeze at −80 °C.
Test efficiency
Transform a known amount of control plasmid (e.g. 100 pg pUC19) and calculate transformants per µg.
Critical points
- Keep cells cold at all times after chilling and handle gently (no vortexing).
- Harvesting past mid-log sharply reduces competence.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Low efficiency | Harvest earlier, keep everything colder, use fresh CaCl₂, use a better strain (e.g. DH5α). |
Safety
BSL-1 lab strains; decontaminate cultures before disposal.
Further reading
- Green MR, Sambrook J. Molecular Cloning (4th edition): preparation of competent E. coli.
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.