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| Product Name | SYBR Green I qPCR Master Mix, 2x, ROX Passive Reference, High Sensitivity |
| Catalog No. | NATR-HMM-0152 |
| Description | Ready-to-use 2x concentrated SYBR Green I-based quantitative real-time PCR master mix containing all components required for qPCR (except primers and template): hot-start Taq DNA polymerase (antibody-mediated), dNTPs (dATP, dCTP, dGTP, dTTP), SYBR Green I fluorescent DNA-binding dye, ROX passive reference dye, MgCl2 (optimized concentration), stabilizers, and PCR buffer. SYBR Green I binds to double-stranded DNA with high affinity, emitting green fluorescence (Ex/Em ~497/520 nm) upon binding, enabling real-time monitoring of PCR product accumulation. The inclusion of ROX passive reference dye normalizes for well-to-well and instrument-to-instrument variation in fluorescence detection, making this master mix compatible with ABI QuantStudio, ABI 7500, and other instruments requiring ROX normalization. The antibody-mediated hot-start polymerase ensures specific amplification with minimal primer-dimer and non-specific product formation. |
| Intended Use | Real-time quantitative PCR for: gene expression analysis by relative quantification (delta-delta Ct method) using SYBR Green detection; DNA quantification (copy number determination); pathogen detection; genotyping by melt curve analysis; ChIP-qPCR; and general qPCR applications not requiring probe-based detection. |
| Principle / Technology | SYBR Green I is a cyanine dye that exhibits minimal fluorescence when free in solution but fluoresces strongly (>1,000-fold enhancement) when bound to the minor groove of double-stranded DNA. As PCR amplification proceeds, the amount of dsDNA product increases proportionally to template copy number, and SYBR Green fluorescence increases in real-time. Fluorescence is measured at each cycle's extension step. The threshold cycle (Ct) is inversely proportional to the log of the initial template concentration. Post-amplification melt curve analysis (60-95 C ramp) distinguishes specific product from primer-dimers and non-specific amplification based on differences in melting temperature (Tm). |
| Detection Method | Prepare 20 uL qPCR reaction: 10 uL 2x Master Mix, 0.2-0.5 uM each primer, 1-100 ng template DNA, water to 20 uL. Thermal cycling: 95 C 3 min (polymerase activation), 40 cycles (95 C 15 s, 60 C 30 s with fluorescence acquisition), followed by melt curve: 60-95 C at 0.5 C increment, 5 s per step with fluorescence acquisition. |
| Sample Type | cDNA (diluted 1:5-1:20 from RT reaction), genomic DNA (1-100 ng per reaction), plasmid DNA (10^2-10^8 copies), microbial DNA. |
| Performance Range / Specifications | Amplification efficiency: 90-110% over 6 orders of magnitude template dilution; linear dynamic range: 10^1-10^8 copies; Ct linearity: R2 >0.99; resolution: reliable discrimination of 2-fold differences in template concentration; ROX concentration: optimized for high-ROX instruments (approximately 500 nM final). |
| Sensitivity / LOD | Detection of <10 copies of target DNA per reaction; single-copy gene detection from 100 pg human genomic DNA; detection of 0.1 pg cDNA from high-abundance transcripts. |
| Specificity | SYBR Green I binds to all double-stranded DNA; specificity of detection determined entirely by primer design. Hot-start antibody-mediated polymerase minimizes non-specific amplification and primer-dimer. Melt curve analysis verifies amplification specificity: specific product yields a single sharp melt peak; primer-dimer yields a broader peak at lower Tm (typically 70-78 C). |
| Reaction Conditions / Protocol | Hot-start activation: 95 C, 3 min; cycling: 95 C 15 s, 60 C 30 s (two-step protocol recommended — or 95 C 15 s, 60 C 30 s, 72 C 30 s three-step for amplicons >200 bp); melt curve: 60-95 C, 0.5 C/5 s. |
| Components / Formulation | 2x SYBR Green I qPCR Master Mix (5 x 1 mL vials): hot-start Taq DNA polymerase (antibody-mediated), dNTPs (0.4 mM each, 0.2 mM each in 1x), SYBR Green I (0.5x final), ROX passive reference dye (optimized concentration), MgCl2 (5 mM in 1x, additional), PCR buffer with stabilizers; nuclease-free water (5 mL, for reaction setup); Protocol. |
| Storage Conditions | Store at -20 C protected from light; stable at 2-8 C for up to 3 months; avoid repeated freeze-thaw cycles (stable for >=20 cycles); do not expose SYBR Green I to light for extended periods. |
| Shelf Life | 24 months from manufacture at -20 C; 3 months at 2-8 C. |
| Package Specifications | 5 x 1 mL (500 reactions at 20 uL volume); also available in 1 mL (100 rxn) and 50 mL (5,000 rxn). |
| Product Form | Orange-red to pink frozen liquid (ROX + SYBR Green); thawed: pink to light orange clear solution. |
| Quality Control | Each lot: PCR efficiency 90-110% (standard curve over 10^1-10^8 copies); linearity R2 >0.99; sensitivity: detection of <=10 copies control template; no-template control (NTC): Ct >35 or no amplification; DNase/RNase: negative; functionally tested with human GAPDH primers. |
| Key Features | 2x master mix — just add primers and template; hot-start antibody-mediated Taq; SYBR Green I with ROX reference; high sensitivity (<10 copies); includes melt curve capability; 24-month shelf life; 500-reaction package. |
| Purity | DNase-free, RNase-free; SYBR Green I: PCR-grade; ROX: PCR-grade; DNA polymerase: >95% pure; host DNA <10 copies per reaction. |
| Concentration | 2x master mix; working concentration after 1:2 dilution: 1x; SYBR Green I at optimized concentration (typically 0.5x final); ROX at optimized concentration for high-ROX instruments. |
| Activity / Unit Definition | Taq DNA polymerase: hot-start antibody-mediated; activity approximately 0.05 U/uL in 1x reaction. |
| Molecular Weight | SYBR Green I: proprietary cyanine dye; ROX (5-carboxy-X-rhodamine): 534.6 g/mol. |
| Source / Origin | Taq polymerase: recombinant, E. coli-expressed; monoclonal anti-Taq antibodies: recombinant; dNTPs: synthetic; SYBR Green I and ROX: synthetic dyes; all components: molecular biology grade; animal-free production. |
| pH Range / Optimal pH | Master mix pH 8.5-9.0 (25 C); Tris-based buffer system; SYBR Green I fluorescence pH dependent — optimal above pH 7.5. |
| Shipping Conditions | Cold pack or dry ice; protect from light; stable at ambient for up to 1 week transit. |
| Expiration Date / Stability | 24 months at -20 C; 3 months at 2-8 C; quality retained after 20 freeze-thaw cycles; protect from light. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. ISO 9001 certified manufacturing. |
| Compatibility | Compatible with all real-time PCR instruments supporting SYBR/FAM channel: ABI 7500/7900HT/QuantStudio series, Bio-Rad CFX96/384/Opus, Roche LightCycler 480/96, Qiagen Rotor-Gene Q, Eppendorf Mastercycler, Analytik Jena qTOWER, and others. ROX concentration optimized for high-ROX instruments (ABI 7500, QuantStudio); for low-ROX instruments (Bio-Rad CFX, Roche LC480), use a low-ROX formulation or this formulation (ROX signal will be higher but acceptable with proper ROX channel calibration). Compatible with standard 96-well, 384-well, and strip-tube formats. Do not use this master mix for probe-based (TaqMan) qPCR — it contains SYBR Green which will interfere with probe detection. |
| Recommended Buffer System | 2x Master Mix: Tris-HCl pH 8.5-9.0, KCl, MgCl2 (3 mM additional, 5 mM total in 1x), dNTPs (0.4 mM each, 0.2 mM each in 1x), hot-start Taq, SYBR Green I, ROX, stabilizers (BSA, glycerol, detergent). |
| Application Notes / Precautions | Upon first thaw, vortex master mix thoroughly and briefly centrifuge to collect contents. SYBR Green I is light-sensitive — keep thawed master mix on ice in the dark and minimize exposure to ambient light. Prepare the reaction master mix (including primers and template) at RT or on ice. For accurate pipetting of small volumes, prepare a master mix of master mix + primers + water, dispense to plate/strips, then add template DNA. Include a no-template control (NTC) in every run to monitor for contamination and primer-dimer. Always perform melt curve analysis after amplification to verify product specificity. For gene expression analysis, normalize Ct values to reference genes (GAPDH, ACTB, 18S rRNA) and calculate relative expression by the delta-delta Ct method. For optimal results, design primers with amplicon size 75-200 bp, Tm 58-62 C, and GC content 40-60%. |
| Batch-to-Batch Consistency | PCR efficiency 90-110% for every lot; NTC Ct >35 or no amplification; sensitivity <=10 copies; linearity R2 >0.99. |
For research use only, not for clinical use.
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