Recommended Reaction

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

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

Quick Protocol (Fast DNA Digestion)

1. 20 µl plasmid DNA: 15 µl H₂O, 2 µl 10× buffer, 1 µg DNA, 1 µl XbaI.
2. 30 µl PCR product: 16 µl H₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl XbaI.
   If unpurified PCR, reduce buffer to 2 µl.
3. 50 µl genomic DNA: 30 µl H₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl XbaI.
4. Mix gently, spin down, incubate at 37 °C for 15–60 min depending on DNA type.
5. (Optional) Heat-inactivate at 80 °C for 20 min.

Double / Multiple Digests

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

Quality Control

• Functional test: Complete λDNA digest in 15 min at 37 °C.
• Star activity: None detected in 3 h at 37 °C; longer incubations may result in star activity.
• Ligation & recutting: >95% ligation at 22 °C, and >95% recutting of ligated fragments.
• Non-specific activity: <10% nicked/linearized plasmid after 4 h.
• Blue/White assay: <1% white colonies, confirming end integrity.

Activity in Different Buffers