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Gel extraction & PCR cleanup

DNA work

Silica column kit-based purification. Use Zymo DNA Clean & Concentrator, NEB Monarch, or Qiagen QIAquick — all follow the same chemistry. Outline below is for kit-style protocols; check the manual for binding/wash volumes.

PCR cleanup (no gel needed)

1. Mix PCR with 5× volumes of binding buffer (DNA-binding solution).
2. Apply to a silica spin column. Centrifuge 30 s at > 10,000 × g. Discard flow-through.
3. Wash with ethanol-containing buffer (200–700 µL depending on kit). Spin 30 s. Discard flow-through. Repeat if specified.
4. Spin empty 1 min to remove residual ethanol.
5. Move column to a clean tube. Elute in 10–30 µL elution buffer or nuclease-free H₂O. Let stand 1 min, then spin 1 min.
6. Quantify on NanoDrop or Qubit.

Gel extraction

1. Run sample on a blue-light-friendly agarose gel (SYBR Safe or GelRed). See agarose gel.
2. Excise the band on a blue-light transilluminator using a clean razor blade. Trim closely — extra agarose dilutes the eluate.
3. Weigh the slice in a 1.5 mL tube. Add 3× volumes of gel solubilisation buffer (e.g., 100 µL/100 mg).
4. Incubate at 50 °C, 5–10 min, vortexing every couple of minutes until fully dissolved.
5. If the kit calls for it, add 1 volume of isopropanol after dissolution (improves recovery for fragments < 500 bp or > 4 kb).
6. Apply to spin column and follow PCR-cleanup steps 2–6 above.

Notes & tips

• Minimise UV exposure; cut on blue light if possible. UV introduces thymine dimers and reduces cloning efficiency.
• Pre-warming elution buffer to 50 °C improves yield by ~10–20%.
• For very small elution volumes (< 10 µL), elute twice in 6 µL each rather than once in 12 µL.
• If your downstream step is salt-sensitive (e.g., electroporation), elute in nuclease-free H₂O, not Tris buffer.