Recommended Reaction

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

Color Buffer includes density reagent plus 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 Esp3I.
2. 30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl Esp3I.
   If PCR is unpurified, use 2 µl 10× buffer.
3. 50 µl (genomic): 30 µl ddH₂O, 5 µl 10× buffer, 10 µl DNA (5 µg), 5 µl Esp3I.
4. Mix gently, quick-spin; incubate at 37 °C for the indicated time.
5. (Optional) Heat-inactivate at 80 °C for 20 min or load gel directly (Color Buffer).

Double / Multiple Digests

• Use ~1 µl of each enzyme; 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: 1 µg λDNA + 1 µl Esp3I 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; >95% of ligated fragments recut by Esp3I.

Activity in Different Buffers

*Activities refer to the functional test conditions.