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| Product Name | Phusion High-Fidelity DNA Polymerase, 2 U/µL |
| Catalog No. | IEC-HMM-0071 |
| Description | Recombinant high-fidelity DNA polymerase with proofreading (3′→5′ exonuclease) activity, engineered through fusion of a dsDNA-binding domain to a Pyrococcus-like proofreading polymerase. This fusion technology dramatically improves processivity and fidelity (error rate ~4.4×10⁻⁷, approximately 50-fold higher fidelity than Taq). Designed for demanding PCR applications requiring accurate amplification of long or GC-rich templates. |
| Intended Use | High-fidelity PCR amplification for cloning, mutagenesis, and sequencing; long-range PCR up to 20 kb genomic DNA; GC-rich template amplification; blunt-end PCR product generation for cloning; site-directed mutagenesis; NGS library amplification. |
| Principle / Technology | Fused dsDNA-binding domain tethers polymerase to template, increasing processivity >10-fold; proofreading 3′→5′ exonuclease activity corrects misincorporation errors in real-time; error rate 50× lower than Taq DNA polymerase |
| Detection Method | Standard 50 µL reaction: 1× Phusion buffer, 200 µM dNTPs, 0.5 µM each primer, template DNA, 0.5-1 U polymerase; typical cycling: 98 °C 30 sec (initial), 98 °C 10 sec / 55-72 °C 30 sec / 72 °C 15-30 sec/kb (30-35 cycles), 72 °C 5-10 min |
| Sample Type | Genomic DNA (10-200 ng), plasmid DNA (1-10 ng), cDNA (1-5 µL RT reaction), bacterial colonies (direct PCR) |
| Sensitivity / LOD | Amplifies from as little as 1 pg plasmid or 10 ng human genomic DNA per 50 µL reaction |
| Specificity | Generates blunt-end products; 3′→5′ exonuclease activity degrades unpaired primers — use carefully designed primers with stable 3′ ends |
| Reaction Conditions / Protocol | Extension rate 15-30 sec/kb for genomic DNA, 15 sec/kb for plasmid; optimal annealing temperature typically Tm +3 °C vs. standard Taq due to dsDNA binding domain |
| Components / Formulation | Phusion DNA Polymerase (2 U/µL), 5× Phusion HF Buffer (with 7.5 mM MgCl₂), 5× Phusion GC Buffer (with 7.5 mM MgCl₂ for GC-rich templates), DMSO (for GC-rich amplification), 50 mM MgCl₂ solution (for Mg²⁺ optimization) |
| Storage Conditions | -20 °C; polymerase in 50% glycerol — does not freeze solid at -20 °C; do not store at -80 °C |
| Shelf Life | 24 months from manufacture date at -20 °C |
| Package Specifications | 100 U, 500 U, 2,500 U; 1 U defined as amount incorporating 10 nmol dNTPs in 30 min at 74 °C |
| Product Form | Liquid enzyme in storage buffer (50% glycerol); clear to slightly opalescent |
| Key Features | 50× higher fidelity than Taq; 10× higher processivity; amplifies up to 20 kb genomic DNA; GC buffer included for GC-rich templates; blunt-end products compatible with blunt-end cloning; DMSO included; rapid extension 15-30 sec/kb |
| Purity | Recombinant, >99% purity SDS-PAGE; no detectable non-specific endonuclease/nickase; endotoxin <0.01 EU/U; host DNA <10 fg/U |
| Concentration | 2 U/µL in 50% glycerol storage buffer |
| Activity / Unit Definition | 2,000 U/mL; unit defined at 74 °C with activated salmon sperm DNA template |
| 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; fusion of Pyrococcus-like DNA polymerase with dsDNA-binding domain |
| 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 | Cold pack -20 °C; enzyme in glycerol — do not freeze to -80 °C |
| Expiration Date / Stability | 24 months at -20 °C; 12 months at -20 °C after first use; keep on ice during benchtop use; return to -20 °C immediately |
| Regulatory / Compliance | For laboratory and research use only; RUO; ISO 9001 |
| Compatibility | PCR products are blunt-ended — compatible with blunt-end cloning vectors; add 3′-A overhang with Taq for TA cloning; GC buffer recommended for templates >65% GC; DMSO (3% final) recommended for GC-rich or structured templates |
| Recommended Buffer System | 5× HF Buffer: 1× provides 1.5 mM MgCl₂; 5× GC Buffer: 1× provides 1.5 mM MgCl₂ + proprietary GC enhancers; Mg²⁺ can be titrated with supplied MgCl₂ solution |
| 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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