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| Product Name | TaqMan Universal qPCR Master Mix, 2x, No UNG, ROX Passive Reference |
| Catalog No. | NATR-HMM-0153 |
| Description | Ready-to-use 2x concentrated TaqMan (hydrolysis probe) quantitative real-time PCR master mix optimized for probe-based detection, containing all components for qPCR except primers, probes, and template: hot-start Taq DNA polymerase (antibody-mediated), dNTPs (dATP, dCTP, dGTP, dTTP — no dUTP), ROX passive reference dye, MgCl2 (optimized), and PCR buffer with stabilizers. This formulation does NOT contain uracil-N-glycosylase (UNG) and uses dTTP instead of dUTP, making it compatible with standard DNA templates and primers without modification of thermocycling protocols for UNG incubation. It is designed for probe-based qPCR applications where carryover prevention (UNG/dUTP system) is not required, such as gene expression analysis, copy number variation, SNP genotyping, and pathogen detection using TaqMan, molecular beacon, or Scorpions probe chemistries. |
| Intended Use | Probe-based real-time quantitative PCR for: gene expression analysis with TaqMan probes; SNP genotyping (TaqMan allelic discrimination); copy number variation (CNV) analysis; microRNA quantification (TaqMan miRNA assays); pathogen detection and viral load quantification; and digital PCR on compatible instruments. Optimized for single-plex and multiplex (up to 4-plex) probe-based detection. |
| Principle / Technology | The master mix contains an antibody-mediated hot-start Taq DNA polymerase that is inactive at room temperature. Upon thermal activation at 95 C (2-10 min), the antibodies are denatured, releasing active polymerase. During PCR, the polymerase extends primers and, upon encountering a hybridized TaqMan probe, cleaves the probe via 5'->3' exonuclease activity, separating the fluorophore from the quencher and generating a fluorescence signal proportional to the amount of amplified target. ROX passive reference provides internal normalization for non-PCR-related fluorescence fluctuations. |
| Detection Method | Prepare 20 uL reaction: 10 uL 2x Master Mix, 0.2-0.9 uM each primer, 0.1-0.25 uM probe, 1-100 ng template DNA, water to 20 uL. Standard cycling: 95 C 10 min (polymerase activation), 40 cycles (95 C 15 s, 60 C 60 s with data collection at 60 C). For fast cycling: 95 C 20 s, 40 cycles (95 C 1 s, 60 C 20 s). |
| Sample Type | cDNA (1-5 uL RT reaction diluted 1:5-1:20), genomic DNA (1-100 ng/rxn), plasmid DNA, viral DNA/cDNA. |
| Performance Range / Specifications | Amplification efficiency: 90-110% over 6 logs template dilution; linear range: 10^1-10^8 copies; Ct linearity: R2 >0.99; probe signal-to-noise: >10:1 (cleaved vs intact probe); multiplex capability: up to 4-plex with properly designed probes. |
| Sensitivity / LOD | Detection of <5 copies target DNA per reaction (single-copy sensitivity); detectable Ct difference of 0.5 cycles between 2-fold template dilutions. |
| Specificity | Probe-based detection provides inherent sequence specificity beyond primer specificity: fluorescent signal only generated when probe hybridizes to target and is cleaved by polymerase. Hot-start polymerase ensures no non-specific amplification during reaction setup. No UNG means dTTP is used (not dUTP); template is natural DNA with thymidine. |
| Reaction Conditions / Protocol | Standard protocol: 95 C 10 min activation (antibody-mediated hot start), 40 cycles (95 C 15 s, 60 C 60 s). Fast protocol: 95 C 20-30 s, 40 cycles (95 C 1-3 s, 60 C 20-30 s). For ABI Fast instruments, use fast protocol; for standard instruments, use standard protocol. |
| Components / Formulation | 2x TaqMan Universal qPCR Master Mix, No UNG (5 x 1 mL vials): hot-start Taq polymerase, dNTPs (0.4 mM each dATP, dCTP, dGTP, dTTP), ROX passive reference, MgCl2 (optimized, 5 mM final in 1x), PCR buffer, stabilizers; nuclease-free water (5 mL); Protocol. |
| Storage Conditions | Store at -20 C protected from light; stable at 2-8 C for up to 3 months; avoid repeated freeze-thaw. |
| Shelf Life | 24 months at -20 C. |
| Package Specifications | 5 x 1 mL (500 reactions at 20 uL); 1 mL (100 rxn); 50 mL (5,000 rxn). |
| Product Form | Colorless to pale pink frozen liquid; thawed: clear solution. |
| Quality Control | Each lot: PCR efficiency 90-110% (FAM-TAMRA probe, 6-log standard curve); linearity R2 >0.99; sensitivity: <=5 copies control template; NTC: no amplification (Ct >40 or undetermined); DNase/RNase: negative; ROX signal stability across 40 cycles; functionally tested with human RNase P TaqMan assay. |
| Key Features | 2x probe-based qPCR master mix; no UNG (standard dTTP); hot-start Taq; ROX reference for normalization; up to 4-plex compatible; 24-month shelf life; 500 reactions. |
| Purity | DNase-free, RNase-free; DNA polymerase >95% pure; dNTPs >99% by HPLC; host genomic DNA <10 copies/reaction. |
| Concentration | 2x master mix; Taq polymerase approximately 0.05 U/uL in 1x; MgCl2 5 mM total in 1x; dNTPs 0.2 mM each in 1x. |
| Activity / Unit Definition | Taq polymerase: hot-start antibody-mediated; probe cleavage activity verified; no detectable UNG or other glycosylase activity. |
| Molecular Weight | Not applicable — mixture; Taq polymerase ~94 kDa. |
| Source / Origin | Recombinant Taq polymerase (E. coli); monoclonal antibodies (recombinant); dNTPs (synthetic); ROX (synthetic); all components: molecular biology grade; animal-free production. |
| pH Range / Optimal pH | pH 8.5-9.0 (25 C); Tris-based buffer; optimal pH for Taq polymerase activity. |
| Shipping Conditions | Cold pack or dry ice; protect from light. |
| Expiration Date / Stability | 24 months at -20 C; 3 months at 2-8 C; 20 freeze-thaw cycles tolerated. |
| Regulatory / Compliance | For research use only; not for diagnostic use. ISO 9001 certified. |
| Compatibility | Compatible with all real-time PCR instruments supporting probe-based detection: ABI 7500/7900HT/QuantStudio, Bio-Rad CFX, Roche LightCycler 480, Qiagen Rotor-Gene Q, and others. ROX concentration optimized for high-ROX instruments (ABI 7500, QuantStudio). Compatible with TaqMan, molecular beacon, Scorpions, and other hydrolysis probe chemistries. For multiplex qPCR, use spectrally distinct fluorophores (FAM, VIC/HEX, NED/TAMRA, Cy5) with appropriate quenchers (BHQ-1, BHQ-2, BHQ-3). This no-UNG formulation uses dTTP — do not use with dUTP-containing primers or UNG carryover prevention protocols. |
| Recommended Buffer System | Tris-HCl pH 8.5-9.0, KCl, MgCl2, dNTPs, hot-start Taq, ROX, stabilizers. |
| Application Notes / Precautions | Vortex master mix thoroughly before use. Prepare master mix reactions at RT. For multiplex qPCR, verify that probe fluorophores are spectrally compatible with instrument detection channels and that primer/probe concentrations are optimized to avoid competition. Standard probe concentration: 100-250 nM final. Standard primer concentration: 200-900 nM each. For difficult templates (GC-rich, secondary structure), add DMSO (2-5%) or betaine (1-2 M). The 10-minute activation step at 95 C is essential for complete antibody release. Do not reduce below 2 min. |
| Batch-to-Batch Consistency | PCR efficiency 90-110%; sensitivity <=5 copies; NTC negative; linearity R2 >0.99. |
For research use only, not for clinical use.
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