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| Product Name | SYBR Green qPCR Master Mix (2x) |
| Catalog No. | NATR-HMM-0115 |
| Description | A 2× concentrated real-time quantitative PCR master mix containing a modified hot-start DNA polymerase, SYBR Green I fluorescent dye, dNTPs, and an optimized buffer system for gene expression analysis by qPCR using intercalating dye detection chemistry. |
| Intended Use | Quantitative real-time PCR gene expression analysis, target sequence detection, and copy number determination in cDNA and genomic DNA templates using SYBR Green I fluorescence detection on standard qPCR instrument platforms. |
| Principle / Technology | Double-stranded DNA-selective binding of SYBR Green I dye produces a >1000-fold fluorescence enhancement upon intercalation into the dsDNA minor groove. Fluorescence signal is measured at the end of each extension step and increases proportionally with the amount of PCR product generated. Post-amplification melt curve analysis confirms amplification specificity by monitoring fluorescence change during controlled thermal denaturation of PCR products. |
| Detection Method | Real-time fluorescence monitoring during PCR (SYBR Green I: excitation ~497 nm, emission ~520 nm); post-amplification dissociation (melt) curve analysis for product specificity verification |
| Sample Type | cDNA synthesized from total RNA or mRNA; genomic DNA for copy number analysis; plasmid DNA for standard curve generation |
| Performance Range / Specifications | Amplicon size: 70–300 bp recommended; linear dynamic range: 6–8 orders of magnitude; PCR efficiency: 90–110% for properly designed assays; inter-assay CV <5% across technical replicates |
| Sensitivity / LOD | Detects <10 copies of target cDNA in a 20 μL reaction; distinguishes 1.5-fold changes in gene expression at >95% confidence (Student's t-test, n=3) for targets with Ct <30 |
| Specificity | Melt curve analysis distinguishes specific product from primer-dimers and non-specific amplicons; single Tm peak per well for validated primer sets; no detectable non-specific amplification products in no-template controls |
| Reaction Conditions / Protocol | Prepare 20 μL reactions: 10 μL SYBR Green master mix, forward/reverse primers (100–300 nM each final), 2 μL template cDNA (or 5–50 ng genomic DNA), nuclease-free water; run on qPCR instrument: 95°C 2–5 min, 40 cycles of (95°C 5–15 sec, 60°C 20–30 sec with fluorescence acquisition), followed by melt curve: 60–95°C ramp at 0.5°C increments; analysis by ΔΔCt method with reference gene normalization |
| Components / Formulation | Modified hot-start DNA polymerase, SYBR Green I dye, dNTPs (dATP, dCTP, dGTP, dTTP/dUTP blend), MgCl2 (2.5–3.0 mM final in 1×), passive reference dye (ROX or fluorescein, instrument-dependent), reaction buffer with KCl and (NH4)2SO4, proprietary stabilizers and enhancers |
| Storage Conditions | –20°C protected from light for long-term storage; stable at 2–8°C protected from light for up to 3 months; avoid repeated freeze-thaw cycles (>15) |
| Shelf Life | 24 months at –20°C from date of manufacture; sensitive to light — store in dark container |
| Package Specifications | 200 reactions (2 × 1 mL, based on 20 μL reaction volume), 500 reactions (5 × 1 mL), 2500 reactions (25 mL) |
| Product Form | Liquid 2× master mix, ready to use |
| Quality Control | Each lot tested: standard curve with 10-fold serial dilution of human genomic DNA (R² ≥0.99, efficiency 90–110%); no amplification in no-template control after 40 cycles; single sharp melt peak for control amplicons; consistent Ct values across triplicate reactions (CV <3%); ROX signal stability verified |
| Key Features | Pre-mixed SYBR Green I eliminates separate dye addition; antibody-mediated hot-start for room-temperature setup; optimized for fast thermal cycling protocols (<40 minutes per run); validated on major qPCR platforms; melt curve analysis included in every run for specificity confirmation |
For research use only, not for clinical use.
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