Recognition & Cleavage
Recognition site: 5′-TCTAGA-3′ (palindromic)
5′ ... T▼C T A G A ... 3′ 3′ ... A G A T C▲T ... 5′
Type: II restriction endonuclease · Common use: cloning, verification, and restriction mapping
XbaI (Restriction Enzyme)
REF: EG15581S
Recommended Reaction
- Buffer: 1× CutOne® or 1× CutOne® Color Buffer
- Incubation: 37 °C
- Typical durations: 15 min (plasmid DNA), 15–30 min (PCR products), 30–60 min (genomic DNA)
CutOne® Color Buffer includes density reagent with tracking dyes for direct gel loading.
Quick Protocol (Fast DNA Digestion)
- 20 µl plasmid DNA: 15 µl H₂O, 2 µl 10× buffer, 1 µg DNA, 1 µl XbaI.
- 30 µl PCR product: 16 µl H₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl XbaI.
If unpurified PCR, reduce buffer to 2 µl. - 50 µl genomic DNA: 30 µl H₂O, 5 µl 10× buffer, 5 µg DNA, 5 µl XbaI.
- Mix gently, spin down, incubate at 37 °C for 15–60 min depending on DNA type.
- (Optional) Heat-inactivate at 80 °C for 20 min.
Double / Multiple Digests
- Use ~1 µl of each enzyme; keep total enzyme ≤10% of volume.
- If enzymes require different temperatures, start with the lower, then add the second enzyme and shift higher.
- Increase incubation if total volume >20 µl.
Quality Control
- Functional test: Complete λDNA digest in 15 min at 37 °C.
- Star activity: None detected in 3 h at 37 °C; longer incubations may result in star activity.
- Ligation & recutting: >95% ligation at 22 °C, and >95% recutting of ligated fragments.
- Non-specific activity: <10% nicked/linearized plasmid after 4 h.
- Blue/White assay: <1% white colonies, confirming end integrity.
Methylation Effects
| Methylase | Effect |
|---|---|
| Dam | Blocked |
| Dcm | No effect |
| CpG | No effect |
| EcoKI | No effect |
| EcoBI | No effect |
Recognition Sites in Common DNAs
| DNA | # Sites | DNA | # Sites |
|---|---|---|---|
| λDNA | 1 | ΦX174 | 0 |
| pBR322 | 0 | pUC57 | 1 |
| pUC18/19 | 1 | SV40 | 0 |
| M13mp18/19 | 1 | Adeno2 | 5 |
Activity in Different Buffers
| Buffer | Activity |
|---|---|
| CutOne® | 100% |
| Thermo FastDigest | 50% |
| NEB rCutSmart™ | 100% |
| Takara QuickCut™ | 100% |
Related Products
Tags: Restriction Enzyme