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| Product Name | Nicking Endonuclease Nt.BstNBI (Recognition Site: 5'-GAGTCNNNNN-3', Top-Strand Nicking) |
| Catalog No. | IVDRM-0166 |
| Description | Nt.BstNBI is a nicking endonuclease that recognizes the sequence 5'-GAGTCNNNNN-3' and cleaves only one strand (the top strand) of a double-stranded DNA substrate, generating a single-stranded nick without cutting the duplex through. With an optimal reaction temperature of 55°C, the enzyme provides robust, site-specific nicking activity for molecular cloning workflows. |
| Intended Use | For molecular cloning applications requiring site-specific nicking of double-stranded DNA. |
| Principle / Technology | Nicking endonuclease: cleaves only one strand of the dsDNA substrate. Nt.BstNBI nicks the top strand four nucleotides downstream of its recognition site (5'-GAGTCNNNN↓N-3'), producing a nick rather than a double-strand break. |
| Sample Type | Double-stranded DNA (dsDNA) substrates. |
| Performance Range / Specifications | Concentration: 10 U/µL. One unit (U) is the amount of enzyme required to completely convert 1 µg of supercoiled pUC19 DNA to the open circular form in 1 hour at 55°C in a 50 µL reaction. |
| Specificity | Recognizes 5'-GAGTCNNNNN-3' and nicks the top strand. Methylation effect: none. |
| Reaction Conditions / Protocol | 1× Nt.BstNBI reaction buffer; incubate at 55°C. Heat inactivation: 80°C for 20 min. |
| Components / Formulation | Nt.BstNBI nicking endonuclease, 10 U/µL; supplied with the recommended reaction buffer. |
| Storage Conditions | Store at -25°C to -15°C. |
| Shelf Life | 2 years when stored at -25°C to -15°C. |
| Package Specifications | 2000 U |
| Concentration | 10 U/µL |
| Activity / Unit Definition | One unit (U) is defined as the amount of enzyme required to completely convert 1 µg of supercoiled pUC19 DNA into the open circular form within 1 hour at 55°C in a 50 µL reaction. |
| Expiration Date / Stability | Stable for 2 years at -25°C to -15°C. |
| Recommended Buffer System | 1× dedicated reaction buffer (supplied). |
| Application Notes / Precautions | Incubate reactions at 55°C for optimal activity. Cleavage is not affected by common DNA methylation. Inactivate the enzyme at 80°C for 20 min before downstream steps. Avoid repeated freeze-thaw cycles; store at -25°C to -15°C. |
| Recognition Site | 5'-GAGTCNNNN↓N-3' 3'-CTCAGNNNNN-5' (Nt.BstNBI cleaves the top strand four nucleotides downstream of the recognition site, as indicated by ↓.) |
| Heat Inactivation | 80°C for 20 min. |
| Methylation Sensitivity | Not affected by methylation. |
For research use only, not for clinical use.
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