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)

CutOne® Color Buffer includes 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 BstBI.
2. 30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl BstBI. 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, 10 µl DNA (5 µg), 5 µl BstBI.
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 directly if Color Buffer was used.

Multiple Digests

• Use ~1 µl of each enzyme; keep 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 BstBI 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 BstBI.
• Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.

Activity in Different Buffers

*Activities per functional test conditions.