Agarose gel electrophoresis (DNA)
Standard size separation of DNA fragments. Choose gel % by expected fragment size.
Gel percent → size resolved
| Agarose % | Resolves (bp) |
|---|---|
| 0.7% | 800–10,000 |
| 1.0% | 500–7,000 |
| 1.5% | 200–4,000 |
| 2.0% | 100–2,000 |
| 3.0% | 50–500 |
Procedure
- Weigh agarose; add to 1× TAE in an Erlenmeyer (e.g., 1.0 g into 100 mL for a 1% gel).
- Microwave 1–2 min, swirling every 30 s, until fully dissolved (no flecks). Caution: superheating — wear gloves, swirl slowly.
- Cool to ~60 °C. Add SYBR Safe (1× final, 1 µL/10 mL gel) or GelRed (2 µL/100 mL). Avoid EtBr unless you have to.
- Pour into a sealed casting tray with a comb. Pop bubbles with a pipette tip.
- Let solidify ~30 min. Submerge in 1× TAE in the gel box.
- Mix samples with 6× loading dye (or use direct-load dyes from your master mix).
- Load: 5–10 µL of PCR product, 0.5–1 µg of plasmid digest. Always run a ladder (e.g., NEB 1 kb Plus).
- Run at 100 V for ~30 min for a small gel; 5 V/cm as a general rule.
- Image on a blue-light or UV transilluminator. Use blue light if you'll cut bands for cloning.
50× TAE buffer (1 L)
- Tris base — 242 g
- Glacial acetic acid — 57.1 mL
- 0.5 M EDTA pH 8.0 — 100 mL
- H₂O to 1 L. Dilute 50× to 1× before use (20 mL into 980 mL water).
Notes
- For tight resolution of close-sized fragments, run longer at lower voltage.
- Don't overload — you'll lose resolution. If a band is too dim, re-run with more, not less, dilution.
- For gel extraction (cloning prep), use blue-light only and minimise exposure time. UV nicks DNA.
- 1× TAE can be reused 2–3 runs before pH drift.