Restriction digest
Cut a plasmid or PCR product at defined sites — for diagnostic verification, traditional cloning, or backbone preparation. Use NEB's NEBcloner to pick the right buffer and double-digest combinations.
Diagnostic digest (10 µL)
| Component | Volume |
|---|---|
| Plasmid DNA (200–500 ng) | 1–2 µL |
| 10× CutSmart / rCutSmart buffer | 1 µL |
| Enzyme 1 (5–20 U/µL) | 0.3 µL |
| Enzyme 2 (if double digest) | 0.3 µL |
| Nuclease-free H₂O | to 10 µL |
Incubate at the enzyme's optimal temperature (usually 37 °C) for 30–60 min. Heat inactivate (e.g., 65 °C, 20 min) if specified by NEB.
Preparative digest for cloning (50 µL)
| Component | Volume |
|---|---|
| Plasmid DNA (1–3 µg) | variable |
| 10× CutSmart buffer | 5 µL |
| Enzyme 1 (HF if available) | 1 µL |
| Enzyme 2 (HF) | 1 µL |
| Nuclease-free H₂O | to 50 µL |
Incubate 1–2 h at 37 °C. For backbone prep, add 1 µL Antarctic phosphatase with its buffer in the last 30 min to dephosphorylate ends and prevent self-ligation.
Procedure
- Set up reaction on ice in the order: water → buffer → DNA → enzyme.
- Mix gently by pipetting; do not vortex enzyme.
- Spin 5 s in a microfuge. Incubate at the enzyme's working temperature.
- Run a 5 µL aliquot on an agarose gel to confirm complete digestion.
- Clean up by column purification or gel extraction before downstream use.
Tips
- Use HF (high-fidelity) versions when available — they all use rCutSmart buffer and have minimal star activity.
- Don't exceed 10% glycerol in the reaction (= 1 µL enzyme per 10 µL reaction is the upper limit, since enzymes are stored in 50% glycerol). Star activity gets worse above this.
- Double digests: check the NEB activity-in-double-digest table — most CutSmart enzymes work together at 100%.