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| Product Name | Hot Start Taq DNA Polymerase, Antibody-Mediated, 5 U/uL |
| Catalog No. | IEC-HMM-0076 |
| Description | Antibody-mediated hot start Taq DNA polymerase for highly specific PCR amplification with room-temperature reaction setup capability. The recombinant Taq polymerase is bound by specific monoclonal antibodies that reversibly block polymerase activity at temperatures below 72 C. During the initial denaturation step (95 C, 2-5 min), the antibodies are irreversibly denatured and released, restoring full polymerase activity. This hot start mechanism eliminates non-specific amplification and primer-dimer formation during reaction setup and the initial temperature ramp, resulting in higher specificity, sensitivity, and yield compared to standard Taq polymerase. Supplied at 5 U/uL in glycerol-containing storage buffer with accompanying 10x PCR buffer (with 15 mM MgCl2) and separate 25 mM MgCl2 solution for optimization. |
| Intended Use | Hot start PCR for applications requiring high specificity including: genomic DNA amplification, colony PCR from bacterial colonies, genotyping, SNP and mutation detection, microsatellite analysis, TA cloning (3'-A overhang addition), and high-throughput PCR screening. Suitable for both conventional thermal cyclers and real-time qPCR instruments with SYBR Green detection. |
| Principle / Technology | Monoclonal anti-Taq antibodies bind to the polymerase active site at ambient temperature, preventing extension of non-specifically annealed primers and primer-dimers. During the 95 C initial denaturation step (2-5 minutes), antibodies are denatured and polymerase activity is restored. Taq polymerase then catalyzes 5'->3' synthesis of DNA complementary to the template strand, adding a non-template 3'-A overhang suitable for TA cloning. |
| Detection Method | Prepare PCR reaction on ice or at RT: 1x PCR buffer, 1.5-2.5 mM MgCl2, 0.2 mM each dNTP, 0.2-0.5 uM each primer, 10-100 ng template DNA, 1.25 U Taq polymerase per 50 uL reaction; initial denaturation 95 C, 2-5 min; 30-40 cycles: 95 C 30 s, 50-65 C 30 s (annealing), 72 C 30-60 s/kb (extension); final extension 72 C 5 min. |
| Sample Type | Genomic DNA (10-100 ng), plasmid DNA (1-10 ng), cDNA (1-5 uL RT reaction), bacterial colony (direct transfer with sterile tip), crude cell lysates. |
| Performance Range / Specifications | Amplification efficiency: 90-110% over 5 orders of magnitude template input; amplicon size: 200 bp to 5 kb (standard), up to 10 kb with optimized conditions; extension rate: approximately 1 kb/min at 72 C; 3'-A overhang addition efficiency >90%. |
| Sensitivity / LOD | Detection of single-copy gene from 100 pg human genomic DNA (approximately 15 diploid genome equivalents); detection of 10 copies of plasmid DNA by end-point PCR. |
| Specificity | Antibody-mediated hot start reduces non-specific amplification and primer-dimer by >95% compared to standard Taq; no detectable polymerase activity at 25 C or 37 C after 24 hours incubation. |
| Reaction Conditions / Protocol | Initial denaturation: 95 C, 2-5 min (full antibody release); cycling denaturation: 95 C, 30 s; annealing: 50-65 C, 30 s (optimize per primer pair); extension: 72 C, 60 s per kb. |
| Components / Formulation | Hot Start Taq DNA Polymerase (5 U/uL, in 50% glycerol storage buffer), 10x PCR Buffer (200 mM Tris-HCl pH 8.4, 500 mM KCl, 15 mM MgCl2), 25 mM MgCl2 Solution, Protocol. |
| Storage Conditions | Store at -20 C; stable at 2-8 C for up to 3 months; avoid repeated freeze-thaw cycles (stable for at least 20 cycles). |
| Shelf Life | 24 months from date of manufacture at -20 C. |
| Package Specifications | 250 U (50 uL), 500 U (100 uL), 1,000 U (200 uL), 5,000 U (1 mL). |
| Product Form | Liquid; 5 U/uL in 50% glycerol (v/v), 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP-40. |
| Quality Control | Each lot tested: no detectable endonuclease or nicking activity (incubation with supercoiled pUC19 plasmid at 37 C for 16 h, >90% remaining supercoiled); no detectable exonuclease activity (incubation with 5'- and 3'-labeled oligonucleotides); PCR specificity and sensitivity confirmed using single-copy human genomic target; hot start function confirmed (no primer-dimer in mock reaction without template after 16 h at RT). |
| Key Features | Antibody-mediated hot start; room-temperature reaction setup; high specificity with no primer-dimer; TA cloning compatible (3'-A overhang); 5 kb standard amplicon; stable at 2-8 C for 3 months. |
| Purity | >95% pure by SDS-PAGE (single band at ~94 kDa); no detectable endonuclease, exonuclease, or nicking activities; host genomic DNA <10 copies per unit of enzyme. |
| Concentration | 5 U/uL (1 U = amount of enzyme incorporating 10 nmol dNTP into acid-insoluble form in 30 min at 74 C using activated salmon sperm DNA as template). |
| Activity / Unit Definition | Specific activity: >=20,000 U/mg protein; functional activity confirmed by PCR amplification of 2 kb target from 10 ng human gDNA. |
| Molecular Weight | ~94 kDa (Taq DNA polymerase, full-length); antibody complex molecular weight variable. |
| Source / Origin | Recombinant Thermus aquaticus DNA polymerase expressed in E. coli; monoclonal antibodies produced in mouse hybridoma cell lines and purified by protein A affinity chromatography; no animal-derived components in final enzyme formulation. |
| pH Range / Optimal pH | Optimal pH 8.3-9.0 at 25 C (Tris-HCl); activity range pH 7.5-10.0; 10x PCR buffer pH 8.4 at 25 C. |
| Shipping Conditions | Cold pack or dry ice; stable at ambient for up to 1 week during transit. |
| Expiration Date / Stability | 24 months at -20 C; 3 months at 2-8 C; quality retained after 20 freeze-thaw cycles. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. ISO 9001 certified manufacturing. |
| Compatibility | Compatible with all standard thermal cyclers; reaction conditions compatible with SYBR Green I and EvaGreen real-time PCR detection; PCR products have 3'-A overhangs for direct TA cloning into pGEM-T, pMD18-T, or pCR2.1 vectors. Compatible with dUTP for carryover prevention (use with UNG). Not recommended for high-fidelity applications (error rate ~2.2x10^-5 errors/base/doubling). |
| Recommended Buffer System | 10x PCR Buffer: 200 mM Tris-HCl pH 8.4, 500 mM KCl, 15 mM MgCl2; enzyme storage: 20 mM Tris-HCl pH 8.0, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% NP-40, 50% glycerol. |
| Application Notes / Precautions | Optimal Mg2+ concentration varies by primer pair and template (range 1.5-3.0 mM final). Include the initial denaturation step (95 C, 2-5 min) for complete antibody release. For bacterial colony PCR, transfer a small amount of colony material with a sterile tip and mix directly into the PCR reaction; a brief 95 C, 5 min pre-incubation lyses bacteria and releases antibody block. For GC-rich templates (>65% GC), add DMSO (2-10% final) or betaine (1-2 M final). This enzyme has standard Taq error rate; use a proofreading polymerase for cloning applications requiring high fidelity. |
| Batch-to-Batch Consistency | PCR specificity and yield within established reference range for control template (2 kb amplicon); hot start function: no non-specific products at 2.5x enzyme amount; activity assay: 5,000 +/-500 U/mL. |
For research use only, not for clinical use.
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