Plasmid miniprep

DNA work

Recovers ~10–30 µg of pure plasmid DNA from a 4–5 mL overnight culture. Outline based on the standard alkaline-lysis silica-column workflow (Zymo, NEB Monarch, Qiagen).

You'll need

4–5 mL overnight bacterial culture (LB + antibiotic, 37 °C, ~16 h)
Miniprep kit (resuspension P1 + lysis P2 + neutralisation P3 + wash + elution buffers)
Silica spin columns & collection tubes
Microcentrifuge (capable of > 10,000 × g)

Procedure

Pellet 1.5–4 mL of overnight culture in a 1.5 or 2 mL tube. 1 min at > 10,000 × g. Aspirate supernatant. (For high yield, repeat with the remaining culture into the same pellet.)
Resuspend in 200 µL P1 (RNase-containing). Vortex until no clumps remain.
Lyse by adding 200 µL P2. Invert gently 4–6 times — do not vortex. Solution becomes clear/blue. Wait ≤ 5 min (any longer shears genomic DNA).
Neutralise with 400 µL P3. Invert 4–6 times. White flocculent precipitate forms.
Centrifuge 5 min at max speed. Carefully transfer supernatant (~750 µL) to a silica column.
Spin 30 s. Discard flow-through.
Wash with kit wash buffer (typically 200–700 µL). Spin 30 s. Discard. Repeat if kit calls for it.
Spin empty 1 min to remove residual ethanol.
Place column in a clean 1.5 mL tube. Add 30–50 µL elution buffer or nuclease-free H₂O directly to the column matrix. Wait 1 min.
Spin 1 min at max speed. Quantify by NanoDrop / Qubit. Expect 50–500 ng/µL depending on plasmid copy number.

Notes

For low-copy plasmids (e.g., pBBR, pSC101, BACs), grow up to 10 mL and use a midiprep kit, or extend overnight to 18–24 h.
OD_260/280 should be ~1.8; OD_260/230 > 2.0 indicates clean DNA. Lower 230 ratio = guanidinium carryover (won't break most downstream steps but can inhibit electroporation).
For sequencing-quality DNA, a single miniprep is usually plenty.
Storage: −20 °C long term. Avoid repeated freeze-thaw — aliquot if you'll use often.