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| Product Name | Klenow Fragment (3′→5′ Exo–) |
| Catalog No. | IEC-HMM-0067 |
| Description | The large fragment of Escherichia coli DNA polymerase I retaining 5′→3′ polymerase activity but lacking both 3′→5′ and 5′→3′ exonuclease functions. This exonuclease-deficient variant is specifically engineered for applications requiring uniform nucleotide incorporation without proofreading or nick translation activity. Primary uses include random primer labeling, filling in 5′-overhangs to create blunt ends, and sequencing reactions requiring homogeneous product lengths. |
| Intended Use | Template-dependent DNA synthesis without proofreading for random-primed probe labeling and fill-in reactions. |
| Principle / Technology | 5′→3′ DNA polymerase activity on single-stranded template with primer; no exonuclease activity |
| Detection Method | Radioactive or fluorescent detection of incorporated labeled nucleotides |
| Sample Type | Linear or nicked double-stranded DNA with recessed 3′-termini; single-stranded DNA with annealed primer |
| Performance Range / Specifications | Incorporation rate: approximately 100 nucleotides per second at 37 °C |
| Sensitivity / LOD | Efficient fill-in from sub-femtomole template quantities |
| Specificity | Lacks 3′→5′ exonuclease proofreading; nucleotide misincorporation rate approximately 1 × 10⁻⁵ |
| Reaction Conditions / Protocol | Labeling: incubate 25 ng template with random primers, Klenow (Exo–), dNTPs including labeled dNTP, 1× buffer; 37 °C for 30 min; stop with EDTA |
| Components / Formulation | Klenow Fragment (3′→5′ Exo–) (5 U/µL), 10× Klenow reaction buffer (500 mM Tris-HCl, 100 mM MgCl₂, 10 mM DTT, pH 7.5) |
| Storage Conditions | –20 °C |
| Shelf Life | 24 months at –20 °C |
| Package Specifications | 500 U, 2500 U |
| Product Form | Liquid enzyme in glycerol storage buffer |
| Quality Control | Gap-filling activity verified by filling dNTP mix into BstNI-digested plasmid; exonuclease absence confirmed by 16 hr incubation with 5′-labeled oligonucleotide |
| Key Features | Lack of both exonuclease activities ensures uniform product length in labeling reactions |
| 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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