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| Product Name | DNase I (RNase-free, Recombinant) |
| Catalog No. | IEC-HMM-0054 |
| Description | A recombinant bovine pancreatic deoxyribonuclease I expressed without animal-derived components and rigorously purified to eliminate RNase activity. This endonuclease non-specifically cleaves single- and double-stranded DNA, producing 5'-phosphorylated oligonucleotides with an average length of 2-4 bases. |
| Intended Use | For removal of genomic DNA contamination from RNA preparations prior to RT-PCR or RNA-seq; footprinting assays; nick translation; actinomycin D-mediated DNA cleavage; and any application requiring non-specific DNA digestion in the presence of RNA. |
| Principle / Technology | DNase I hydrolyzes phosphodiester bonds in the DNA backbone through a mechanism involving a histidine residue in the active site, coordinated with divalent cations (Mg²⁺ plus Ca²⁺ or Mn²⁺). Calcium ions stabilize the enzyme structure, while magnesium or manganese facilitates catalysis. |
| Detection Method | One Kunitz unit produces an increase in A₂₆₀ of 0.001 per minute per mL at 25 °C (pH 5.0) using highly polymerized DNA substrate under defined assay conditions. |
| Sample Type | RNA solutions containing contaminating genomic DNA. Typical treatment: 1-2 U DNase I per ug RNA in 10-50 uL total volume. |
| Performance Range / Specifications | Digests single- and double-stranded DNA to oligonucleotides of 2-4 bases average length. Activity in standard RNA buffers (Tris, HEPES, phosphate) containing Mg²⁺. |
| Sensitivity / LOD | Removes detectable DNA contamination from up to 10 ug RNA per 50 uL reaction in 30 min at 37 °C. |
| Specificity | Specific for DNA substrates. Does not degrade RNA when used under recommended conditions with Mg²⁺ as sole divalent cation cofactor. |
| Reaction Conditions / Protocol | Add 1-2 U DNase I per ug RNA in appropriate buffer with 2.5 mM MgCl₂. Incubate 37 °C, 15-30 min. Inactivate with EDTA (5 mM final) and heat at 65 °C 10 min, or use phenol-chloroform extraction. |
| Components / Formulation | Recombinant DNase I (RNase-free, 2 U/uL) in 10 mM Tris-HCl (pH 7.5), 50% glycerol, 10 mM CaCl₂. Supplied with 10x DNase I Reaction Buffer. |
| Storage Conditions | Store at -20 °C to -30 °C. Do not vortex; invert gently to mix. |
| Shelf Life | 24 months at -20 °C. |
| Package Specifications | 2,000 U (1 mL), 10,000 U (5 mL), 50,000 U (25 mL). Includes 10x Reaction Buffer. |
| Product Form | Clear, colorless to pale yellow glycerol-based liquid. Handle gently; avoid foaming. |
| Quality Control | Each lot tested for: DNase specific activity; RNase absence (verified by incubation with MS2 RNA followed by RT-qPCR or gel analysis); absence of non-specific phosphatase and protease activities. |
| Key Features | Recombinant production eliminates animal-derived components; rigorously RNase-free; works efficiently in a wide range of buffer conditions; compatible with downstream RT-PCR after heat inactivation. |
| Purity | >= 95% by SDS-PAGE. No detectable RNase activity after 16-hour incubation with MS2 RNA substrate. |
| Concentration | 2 U/uL standard. Higher concentration variants available for bulk processing. |
| Activity / Unit Definition | 1 Kunitz unit = increase in A₂₆₀ of 0.001/min/mL at 25 °C, pH 5.0. |
| Molecular Weight | ~31 kDa (monomer glycoprotein). |
| Source / Origin | Recombinant expression in Pichia pastoris or E. coli using synthetic gene encoding bovine DNase I sequence. Animal-origin-free manufacturing. |
| pH Range / Optimal pH | Optimal activity at pH 7.0-8.0 (dependent on divalent cation type). Active range pH 5.0-9.0. |
| Shipping Conditions | Shipped with cold packs. Do not freeze above -20 °C; sensitive to shear forces. |
| Expiration Date / Stability | 24 months at -20 °C. Sensitive to physical shearing; handle gently to maintain activity. |
| Regulatory / Compliance | ISO 9001 certified manufacturing. Animal-origin-free process suitable for IVD applications. |
| Compatibility | Compatible with common RNA purification buffers and subsequent RT-PCR, qPCR, and RNA-seq library preparation. Heat inactivation at 65 °C with EDTA (5 mM) is sufficient for most downstream applications. |
| Recommended Buffer System | 10x Reaction Buffer: 100 mM Tris-HCl (pH 7.5), 25 mM MgCl₂, 5 mM CaCl₂. EDTA-free to maintain divalent cation availability for enzyme activity. |
| Application Notes / Precautions | Always include Ca²⁺ (1-5 mM) for enzyme stability during storage and reaction setup. Use Mg²⁺ alone for catalysis. For RNA-seq applications, ensure complete inactivation (phenol-chloroform extraction recommended). Do not heat-inactivate without EDTA; Mg²⁺-catalyzed RNA hydrolysis may occur at elevated temperatures. |
| Batch-to-Batch Consistency | Lot-to-lot DNase activity variation <= +/- 10%. RNase absence re-verified for each production batch by highly sensitive fluorescent substrate assay. |
For research use only, not for clinical use.
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