Recommended Reaction

• Buffer: 1× CutOne® or 1× CutOne® Color Buffer
• Incubation: 37 °C
• Typical durations: 15 min (plasmid), 15–30 min (PCR), 30–60 min (genomic DNA)

CutOne® Color Buffer includes density reagent + tracking dyes for direct gel loading.

Quick Protocol (Fast DNA Digestion)

1. 20 µl plasmid DNA: 15 µl ddH₂O, 2 µl 10× buffer, ≤ 1 µg DNA, 1 µl NheI.
2. 30 µl PCR product: 16 µl ddH₂O, 3 µl 10× buffer, ∼ 0.2 µg DNA, 1 µl NheI.
   If PCR is unpurified, reduce buffer to 2 µl due to residual metal ions.
3. 50 µl genomic DNA: 30 µl ddH₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl NheI.
4. Mix gently, quick-spin; incubate at 37 °C for the indicated time.
5. (Optional) Heat-inactivate at 65–80 °C for 20 min; direct gel loading is possible with Color Buffer.

Double / Multiple Digests

• Use ~1 µl of each enzyme; keep total enzyme volume ≤ 10% of reaction.
• If temperatures differ, start at the lower temperature, then add the second enzyme and shift higher.
• Increase incubation time if total volume > 20 µl.

Quality Control

• Functional test: Complete digest of λ DNA (contains an NheI site) in ≤ 30 min at 37 °C under recommended conditions.
• Prolonged incubation / star activity: 3 h at 37 °C shows no non-specific degradation under recommended conditions.
• Ligation & recutting: After over-digest, > 95% ligation at 22 °C; > 95% recutting of ligated fragments.
• Non-specific endonuclease: < 10% nicked/linearized plasmid after 4 h at 37 °C.

Activity in Different Buffers

Values are guideline-level under the stated test conditions.