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| Product Name | T4 Polynucleotide Kinase |
| Catalog No. | IEC-HMM-0066 |
| Description | A bifunctional enzyme purified from Escherichia coli expressing the cloned bacteriophage T4 polynucleotide kinase gene. The enzyme catalyzes both the transfer of the gamma-phosphate from ATP to the 5′-hydroxyl terminus of DNA or RNA (forward reaction) and the exchange of 5′-terminal phosphate groups with ATP (exchange reaction). Essential applications include radioactive or fluorescent 5′-end labeling of oligonucleotides and phosphorylation of PCR primers prior to ligation into dephosphorylated vectors. |
| Intended Use | 5′-phosphorylation of DNA and RNA oligonucleotides for labeling, ligation, and cloning workflows. |
| Principle / Technology | ATP-dependent 5′-hydroxyl kinase activity and 5′-phosphatase exchange activity |
| Detection Method | Downstream: autoradiography (for ³²P-labeled), fluorescence imaging, or ligation efficiency assessment |
| Sample Type | Single-stranded or double-stranded DNA, RNA, oligonucleotides with 5′-hydroxyl or 5′-phosphate termini |
| Performance Range / Specifications | Phosphorylation: 1 unit phosphorylates 1 nmol 5′-OH termini in 30 min at 37 °C |
| Sensitivity / LOD | Effective labeling from sub-picomole quantities of oligonucleotide |
| Specificity | Active on both DNA and RNA substrates; 5′-overhang, blunt, and recessed ends all phosphorylated with similar efficiency |
| Reaction Conditions / Protocol | Forward reaction: incubate DNA (1–50 pmol) with 1× PNK buffer, 1 mM ATP, 10 units PNK at 37 °C for 30 min; heat inactivate at 65 °C for 20 min |
| Components / Formulation | T4 Polynucleotide Kinase (10 U/µL), 10× PNK reaction buffer (700 mM Tris-HCl, 100 mM MgCl₂, 50 mM DTT, pH 7.6), ATP (10 mM) |
| Storage Conditions | –20 °C |
| Shelf Life | 24 months at –20 °C |
| Package Specifications | 500 U, 2500 U |
| Product Form | Liquid enzyme in storage buffer (50% glycerol) |
| Quality Control | Phosphatase and nuclease contamination: none detected; ligation efficiency of phosphorylated product ≥90% in T4 DNA ligase control test |
| Key Features | Accepts both DNA and RNA substrates with equivalent catalytic efficiency |
| Purity | ≥95% by SDS-PAGE for enzymes; ≥98% by HPLC for coenzymes |
| Concentration | Enzymes at specified U/µL; coenzymes at specified mM concentration |
| Activity / Unit Definition | One unit incorporates 10 nmol dNTP into acid-insoluble form in 30 min at specified temperature (polymerases); other enzyme units per product specification |
| Molecular Weight | As specified per enzyme: Taq ~94 kDa; M-MLV RT ~75 kDa; T4 ligase ~55 kDa; coenzymes as labeled |
| Source / Origin | Recombinant, expressed in E. coli; purified by multi-step chromatography |
| pH Range / Optimal pH | pH 7.5–8.5 for polymerase activity; pH 7.5–8.0 for ligation; pH 7.5–8.0 for transcription |
| Shipping Conditions | Dry ice for enzyme shipments; cold packs for coenzyme solutions |
| Expiration Date / Stability | 24 months at –20 °C for enzymes; 12–36 months for coenzymes per product type |
| Regulatory / Compliance | Research use; ISO 9001 manufacturing; GMP-grade options available for diagnostic manufacturing |
| Compatibility | Compatible with standard PCR, RT-PCR, isothermal amplification, and molecular cloning workflows |
| Recommended Buffer System | Tris-HCl, KCl, MgCl₂-based buffers; glycerol-containing storage buffers |
| Application Notes / Precautions | Aliquot enzymes to avoid repeated freeze-thaw; use dedicated nuclease-free workspace; verify enzyme activity for critical applications |
| Batch-to-Batch Consistency | Enzyme specific activity within ±15% of reference lot; functional testing per lot |
For research use only, not for clinical use.
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