Before you start

Pre-warm selective plates to room temperature/37 °C and set the water bath to exactly 42 °C.

Reagents and materials

Totals are for 4 transformations (+10% extra). Per-transformation amounts are in the middle column.

Reagent	Per transformation	Total
Chemically competent cells	50 µL	50 µL
Plasmid DNA (1–100 ng) or ligation (≤ 10% of cell volume)	2 µL	2 µL
SOC medium	250 µL	250 µL
Selective LB agar plate	1 each	1 each

Tryptone	20 g
Yeast extract	5 g
NaCl	0.584 g (10 mM)
KCl	0.186 g (2.5 mM)
After autoclaving: 1 M MgCl₂ + 1 M MgSO₄ (10 mM each)	10 mL each
After autoclaving: 1 M glucose, filter-sterilised (20 mM)	20 mL

SOB is SOC without glucose.

Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.

Thaw cells on ice
Thaw competent cells on ice (about 10 min). Do not warm by hand.
Add DNA
Add DNA, flick gently to mix (no pipetting up and down) and keep on ice for 30 min.
Heat shock
Place tubes at 42 °C for 30–45 s exactly (per the cells’ instructions).
Back on ice
Return to ice for 2 min.
Recover
Add 250–950 µL SOC and shake at 37 °C for 1 h.
Plate
Spread 50–200 µL on selective plates (for ligations, also plate a concentrated pellet).
Incubate
Incubate inverted at 37 °C overnight (16–18 h).

Controls: uncut vector (efficiency), no DNA (contamination/antibiotic check) and vector-only ligation (background).
Recovery can be shortened for ampicillin selection, but is needed for kanamycin and others.

Problem	What to check or change
No colonies	Check competent-cell efficiency with a control plasmid, antibiotic choice and concentration, heat-shock timing.
Lawn or many satellites	Old ampicillin plates or too long incubation; use fresh plates or carbenicillin.

BSL-1 for lab E. coli strains; decontaminate cultures before disposal.