Recommended Reaction

• Buffer: 1× CutOne® or Color Buffer
• Incubation: 37 °C
• 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 SpeI
2. 30 µl PCR product: 16 µl H₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl SpeI
   If PCR unpurified, reduce buffer to 2 µl
3. 50 µl genomic DNA: 30 µl H₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl SpeI
4. Mix gently, spin down, incubate at 37 °C for indicated times
5. (Optional) Heat-inactivate at 80 °C for 20 min; direct load possible with Color Buffer

Double / Multiple Digests

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

Quality Control

• Functional test: pUC19-SpeI DNA fully digested in 15 min at 37 °C
• Star activity: No nuclease degradation after 3 h; longer incubations may show star activity
• Ligation & recutting: >95% ligation at 22 °C; >95% recutting of ligated DNA
• Non-specific activity: <10% nicked/linearized plasmid after 4 h at 37 °C
• Blue/White assay: <1% white colonies, confirming high end integrity

Activity in Different Buffers