Transformation is the uptake and replication of foreign DNA by bacterial cells. Most laboratory strains of Escherichia coli are not naturally competent — they must be artificially induced to take up DNA.

Competent Cell Preparation

Chemically competent cells are prepared by washing log-phase E. coli with ice-cold CaCl₂ (50–100 mM). The calcium ions neutralize the negatively charged phosphate backbone of DNA and the lipopolysaccharide layer of the bacterial outer membrane, facilitating DNA binding. The cells are then aliquoted and frozen at −80 °C.

Electrocompetent cells are washed repeatedly in ice-cold 10% glycerol to remove all ions from the suspension. The low ionic strength prevents arcing during electroporation. Cells are resuspended in a small volume of 10% glycerol, aliquoted, and frozen.

Chemical Transformation Protocol

1. Thaw a tube of chemically competent cells on ice (about 20 minutes).
2. Add 1–10 µL of DNA (0.1–100 ng of plasmid, or up to 5 µL of a ligation reaction).
3. Incubate on ice for 20–30 minutes.
4. Heat shock at 42 °C for 45 seconds (exactly — longer reduces viability).
5. Return to ice for 2 minutes.
6. Add 500–900 µL of SOC or LB medium (no antibiotic).
7. Incubate at 37 °C with shaking at 200–225 rpm for 45–60 minutes.
8. Spread 50–200 µL onto selective agar plates.
9. Incubate inverted overnight at 37 °C.

Typical efficiency: 10⁶–10⁸ CFU/µg for chemically competent cells.

Electroporation Protocol

1. Thaw electrocompetent cells on ice.
2. Transfer cells to a chilled 0.1 or 0.2 cm cuvette.
3. Add 1–2 µL of DNA in low-salt buffer or water (salt causes arcing).
4. Electroporate at 1.5–2.5 kV (depending on cuvette gap), 200–400 Ω, 25 µF.
5. Immediately add 500 µL of SOC medium and resuspend.
6. Transfer to a culture tube and incubate at 37 °C for 45–60 minutes.
7. Plate on selective agar.

Typical efficiency: 10⁹–10¹⁰ CFU/µg for small plasmids. Electroporation is preferred for large constructs (>10 kb) and ligation products.

Selection

Transformed cells are selected on agar containing an antibiotic matching the resistance marker on the plasmid. Common antibiotics: ampicillin (100 µg/mL), kanamycin (50 µg/mL), chloramphenicol (25 µg/mL), tetracycline (12.5 µg/mL). Ampicillin selection requires fresh plates because the β-lactamase diffuses and degrades the antibiotic — satellite colonies appear on old plates.