Recommended Reaction

• Buffer: 1× CutOne® or 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 red/yellow 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 SphI
2. 30 µl PCR product: 16 µl H₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl SphI (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 SphI
4. Incubate at 37 °C for recommended time
5. Optional: Heat-inactivate at 80 °C for 20 min, or load directly if Color Buffer used

Double / Multiple Digests

• Use ~1 µl of each enzyme; total enzyme ≤10% of volume
• If different temps required, start lower then add enzyme for higher temp
• Increase incubation if total reaction >20 µl

Quality Control

• Functional test: λDNA fully digested in 15 min at 37 °C
• Star activity: None detected at 3 h; longer incubation may result in star activity
• Ligation & recutting: >95% ligation, >95% recutting after over-digest
• Non-specific nuclease: <10% nicked/linearized plasmid after 4 h
• Blue/White assay: <1% white colonies

Activity in Different Buffers