Recommended Reaction

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

CutOne® Color Buffer contains density reagent with tracking dyes for direct gel loading.

Quick Protocol (Fast DNA Digestion)

1. 20 µl plasmid DNA: 15 µl H₂O, 2 µl 10× buffer, ≤1 µg DNA, 1 µl PstI.
2. 30 µl PCR product: 16 µl H₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl PstI.
   If PCR is unpurified, use 2 µl 10× buffer due to residual ions.
3. 50 µl genomic DNA: 30 µl H₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl PstI.
4. Mix gently, quick-spin, incubate at 37 °C for the indicated time.
5. Note: PstI cannot be heat-inactivated; purify DNA after digestion (spin column or phenol/chloroform).

Double / Multiple Digests

• Use ~1 µl of each enzyme; keep total enzyme ≤10% of reaction volume.
• If temperatures differ, start at the lower temperature, then add the second enzyme and shift higher.
• Increase incubation if total volume >20 µl.

Quality Control

• Functional test: Complete digest in 15 min at 37 °C under fast-digest conditions.
• Prolonged incubation / star activity: Stable for 3 h; excessive incubation may cause star activity.
• Ligation & recutting: >95% ligation at 22 °C; >95% of ligated fragments recut by PstI.
• Non-specific nuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.

Activity in Different Buffers