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| Product Name | TaqMan Probe qPCR Master Mix (2x) |
| Catalog No. | NATR-HMM-0116 |
| Description | A 2× concentrated real-time quantitative PCR master mix formulated for probe-based detection using TaqMan hydrolysis probe chemistry. It includes a highly processive hot-start DNA polymerase with 5ʹ→3ʹ exonuclease activity, dNTPs with dUTP, and an optimized buffer system for sensitive and specific target detection. |
| Intended Use | Quantitative and qualitative detection of DNA and cDNA targets using TaqMan probe-based chemistry in gene expression analysis, pathogen detection, SNP genotyping, copy number variation analysis, and miRNA quantification. |
| Principle / Technology | In addition to forward and reverse primers, a dual-labeled oligonucleotide probe anneals to the target sequence between the primers. The probe carries a 5ʹ fluorescent reporter (FAM, VIC, HEX, etc.) and a 3ʹ quencher. During extension, the 5ʹ→3ʹ exonuclease activity of DNA polymerase cleaves the probe, separating reporter from quencher, resulting in fluorescence signal proportional to the amount of amplified product. |
| Detection Method | Real-time fluorescence monitoring on qPCR instrument; multiplex detection with spectrally distinct reporter dyes; endpoint data collection for genotyping applications |
| Sample Type | cDNA from reverse-transcribed RNA; genomic DNA; plasmid DNA controls; viral and bacterial nucleic acid extracts |
| Performance Range / Specifications | Amplicon size range: 50–200 bp; dynamic range: 7–9 logs; PCR efficiency: 95–105% for validated probe-primer sets; multiplex capability: up to 5 targets in a single reaction with appropriate dye selection |
| Sensitivity / LOD | Detection limit: <5 copies of target per 20 μL reaction with single-plex TaqMan assay; 1.2-fold discrimination threshold for gene expression differences between treatment groups |
| Specificity | Probe-based detection provides sequence-specific signal generation; no signal from non-specific amplification or primer-dimers; negligible cross-reactivity between multiplexed probes |
| Reaction Conditions / Protocol | Prepare 20 μL reaction: 10 μL probe master mix, primers (200–900 nM each final), probe (100–250 nM final), template DNA/cDNA, nuclease-free water; standard cycling: 50°C 2 min (UNG incubation for carryover prevention), 95°C 2–10 min (polymerase activation), 40 cycles of (95°C 10–15 sec, 60°C 30–60 sec with fluorescence acquisition); optional 50°C pre-read step for genotyping |
| Components / Formulation | Hot-start DNA polymerase with 5ʹ→3ʹ exonuclease activity, dNTPs with dUTP (replaces dTTP), uracil-N-glycosylase (UNG) for carryover prevention, MgCl2 (optimized), passive reference dye (ROX), proprietary buffer system, stabilizers |
| Storage Conditions | –20°C protected from light; stable at 2–8°C for up to 6 months protected from light; avoid exposure to strong light; limit freeze-thaw cycles to 20 |
| Shelf Life | 24 months at –20°C from date of manufacture |
| Package Specifications | 200 reactions (2 × 1 mL), 500 reactions (5 × 1 mL), 2500 reactions (25 mL), bulk packaging for high-throughput labs |
| Product Form | Liquid 2× master mix |
| Quality Control | Each lot tested: single-copy RNase P gene detection in human genomic DNA (Ct <35 from 5 ng input); multiplex amplification validated with FAM/VIC/Jun-labeled probes; no amplification in no-template controls; UNG carryover prevention validated by dU-containing amplicon challenge; linear dynamic range confirmed 10^-1 to 10^6 copies |
| Key Features | UNG/dUTP carryover prevention system eliminates PCR contamination risk; multiplex-optimized buffer enables up to 5-plex reactions; fast cycling capability (<40 minutes); room-temperature setup stability with hot-start polymerase; compatible with all major qPCR and digital PCR platforms |
For research use only, not for clinical use.
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