Recommended Reaction

• Buffer: 1× CutOne® or 1× CutOne® Color Buffer
• Incubation: 37 °C
• Typical duration: 5–15 min (plasmid/PCR); 30–60 min (genomic DNA)

CutOne® Color Buffer includes density reagent + 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 BsaI.
2. 30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl BsaI.
   If unpurified PCR, reduce buffer to 2 µl due to residual ions.
3. 50 µl (genomic): 30 µl ddH₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl BsaI.
4. Mix gently, spin down; incubate at 37 °C for indicated time.
5. (Optional) Heat-inactivate at 80 °C for 20 min or load gel directly (Color Buffer).

Notes

• Enzyme volume ≤10% of reaction to avoid glycerol-induced star activity.
• Contaminants (phenol, ethanol, EDTA, detergents, high salt) may inhibit digestion.
• Scale-up: for 1–5 µg DNA, increase BsaI and buffer proportionally; extend incubation if volume >20 µl.

Quality Control

• Functional test: Complete digestion of pPIC9K DNA (1 µg) in 15 min at 37 °C with 1 µl enzyme.
• Prolonged incubation: 3 h at 37 °C shows no nuclease degradation; longer may cause star activity.
• Ligation & recutting: >95% ligation with T4 DNA Ligase; >95% of ligated products recut by BsaI.
• Non-specific nuclease: <10% nicking/linearization after 4 h at 37 °C.

Buffer Compatibility (Activity)

*Activities per functional test conditions.