Recommended Reaction

• Buffer: 1× CutOne® or 1× CutOne® Color Buffer
• Incubation: 37 °C
• Guideline: Use fast-digest setup below; scale volumes as needed.

CutOne® Color Buffer contains density reagent with red/yellow tracking dyes for direct gel loading.

Quick Protocol (Fast DNA Digestion)

1. 20 µl (plasmid): 15 µl ddH₂O, 2 µl 10× buffer, ≤1 µg DNA, 1 µl NotI.
2. 30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl NotI.
   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 NotI.
4. Mix gently, quick-spin; incubate at 37 °C for 15 min (plasmid), 15–30 min (PCR), or 30–60 min (genomic).
5. (Optional) Heat-inactivate at 80 °C for 20 min; load directly if Color Buffer was used.

Double / Multiple Digests

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

Quality Control

• Functional test: 1 µg p615 DNA + 1 µl NotI in 20 µl CutOne® → complete digest in 15 min at 37 °C.
• Prolonged incubation / star activity: 3 h at 37 °C shows no nuclease degradation; longer incubation may cause star activity.
• Ligation & recutting: After 10× over-digest, >95% ligation at 22 °C and >95% recutting of ligated fragments.
• Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.
• Blue/White assay: <1% white colonies, confirming end integrity.

Activity in Different Buffers

Activities per functional test conditions.