Gel Extraction and PCR Purification

After PCR amplification or restriction digestion, the DNA must be purified away from enzymes, primers, nucleotides, salts, and buffer components before downstream applications. Two related methods cover most purification needs.

PCR Purification

PCR purification removes primers, dNTPs, polymerases, and salts from an amplification reaction. Most commercial kits use a silica membrane in a spin column: the DNA binds to the membrane in the presence of a high concentration of chaotropic salts, contaminants flow through, and the DNA is eluted in a low-salt buffer (typically 10 mM Tris or water).

The typical protocol: add binding buffer (containing guanidine hydrochloride or similar chaotrope) directly to the PCR reaction, load onto the column, centrifuge, wash with ethanol-based wash buffer, centrifuge again, and elute in 20–50 µL. Recovery is typically 60–90%.

PCR purification is fast (5–10 minutes) and works for fragments >100 bp. It does not remove primer-dimers or non-specific products of similar size — only excess primers and small fragments that pass through the column.

Gel Extraction

Gel extraction isolates a specific DNA band from an agarose gel, removing all other DNA fragments, including primer-dimers and misprimed products. The desired band is excised under UV light using a clean scalpel, and the agarose is dissolved in a chaotropic salt solution at 50–60 °C. The melted agarose is then passed through a silica spin column as in PCR purification.

Key considerations:

Minimize UV exposure time to prevent DNA damage (use 365 nm UV instead of 302 nm if available).
Cut as close to the band as possible to minimize agarose volume.
The dissolution step requires enough buffer — typically 3 volumes per volume of gel.
Yield decreases for fragments >10 kb; incubating the dissolved gel at room temperature instead of on-column improves recovery of large fragments.

DNA Cleanup by Precipitation

Ethanol precipitation is a traditional alternative that does not require kits. Add 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of cold 100% ethanol, incubate at −20 °C or −80 °C, centrifuge at maximum speed for 15–30 minutes, wash with 70% ethanol, air-dry, and resuspend. This method works for any fragment size but takes longer and does not remove dNTPs as efficiently as silica columns.

Quality Control

After purification, measure the A₂₆₀/A₂₈₀ ratio to check purity (ideally 1.8–2.0). Run an aliquot on a gel to verify the band is intact and the correct size. For cloning, the elution buffer should be compatible with downstream enzymes — elute in water or 10 mM Tris (not EDTA if ligation follows).