Before you start

Grow a single colony overnight (12–16 h) in 3–5 mL selective LB at 37 °C with shaking.

Reagents and materials

Totals are for 6 cultures (+10% extra). Per-culture amounts are in the middle column.

Reagent	Per culture	Total
Overnight culture in LB + antibiotic	3 mL	3 mL
Resuspension buffer with RNase A (P1)	250 µL	250 µL
Lysis buffer (P2: NaOH/SDS)	250 µL	250 µL
Neutralisation buffer (N3)	350 µL	350 µL
Wash buffer(s) (per kit)	1.25 mL	1.25 mL
Elution buffer or water	50 µL	50 µL

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Pellet bacteria
Centrifuge 1–5 mL culture at >8,000 × g for 2–3 min and discard the medium.
Resuspend
Resuspend completely in 250 µL P1 (no clumps).
Lyse
Add 250 µL P2 and invert 4–6 times; do not vortex and do not exceed 5 min.
Neutralise
Add 350 µL N3 and invert immediately until a white precipitate forms.
Clear the lysate
Centrifuge at maximum speed for 10 min.
Bind
Apply the supernatant to the column, centrifuge 1 min and discard flow-through.
Wash
Wash as the kit says; spin an extra 1 min to remove residual ethanol.
Elute
Add 30–50 µL elution buffer to the membrane, wait 1 min and centrifuge.
Quantify
Measure concentration and purity (A260/280 ≈ 1.8).

Problem	What to check or change
Low yield	Use fresh cultures, a larger volume, or a high-copy origin; check antibiotic.
Genomic DNA contamination	Do not vortex after lysis; keep lysis under 5 min.

E. coli lab strains: BSL-1. NaOH/SDS buffer is an irritant.