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 AvrII.
2. 30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl AvrII.
   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 AvrII.
4. Mix gently, spin down; incubate at 37 °C for indicated time.
5. Post-digest: Purify the reaction (phenol/chloroform or column) before ligation/transformation.

Notes

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

Quality Control

• Functional test: Complete λDNA digestion (HindIII digest) 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 fragments recut by AvrII.
• Non-specific nuclease: <10% nicking/linearization after 4 h at 37 °C.
• Blue/White assay: <1% white colonies (DNA ends remain intact).

Buffer Compatibility (Activity)

*Activities per functional test conditions.