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× CutOne®, ≤1 µg DNA, 1 µl EcoRI.
2. 30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× CutOne®, ~0.2 µg DNA, 1 µl EcoRI.
   If unpurified PCR, use 2 µl 10× buffer.
3. 50 µl (genomic): 30 µl ddH₂O, 5 µl 10× CutOne®, 5 µg DNA, 5 µl EcoRI.
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).

Notes

• Total enzyme volume ≤10% of reaction to avoid glycerol-induced star activity.
• Contaminants (phenol, ethanol, EDTA, high salt, detergents) inhibit digestion—purify DNA if needed.
• Increase incubation time if total volume >20 µl.

Quality Control

• Functional test: Complete λDNA digestion in 15 min at 37 °C (20 µl, 1 µg DNA, 1 µl enzyme).
• Prolonged incubation: 3 h at 37 °C shows no nuclease degradation; longer may cause star activity.
• Ligation & recutting: >95% ligation at 22 °C (T4 DNA ligase); >95% of ligated products recut by EcoRI.
• Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.
• Blue/White assay: <1% white colonies (DNA ends remain intact).

Buffer Compatibility (Activity)

*Activities per functional test conditions.