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| Product Name | qPCR SYBR Green Master Mix (High ROX Plus) |
| Catalog No. | RESK-0009 |
| Description | qPCR SYBR Green Master Mix (High ROX Plus) is a 2× ready-to-use premix for real-time quantitative PCR. The mix contains hot-start DNA polymerase, SYBR Green I, dNTPs, Mg2+, and a High ROX reference dye; only template and primers need to be added, which streamlines setup and minimizes the risk of contamination. The DNA polymerase ligand modulates enzyme activity in real time as temperature changes, and the formulation incorporates factors that suppress nonspecific amplification and enhance amplification efficiency, delivering excellent linearity across a broad quantification range. |
| Intended Use | Real-time quantitative PCR (dye-based) for gene expression analysis and nucleic acid quantification on qPCR instruments requiring a High ROX reference dye. For research use. |
| Principle / Technology | SYBR Green I dye-based detection: the dye fluoresces upon binding double-stranded DNA, so signal intensity tracks amplicon accumulation in real time. The hot-start DNA polymerase is regulated by a temperature-sensitive ligand (ligand-based hot start) that keeps the enzyme inactive at low temperatures and releases activity during thermal cycling, reducing nonspecific priming. The optimized buffer includes enhancers that suppress nonspecific amplification and improve amplification efficiency across templates with 30%-70% GC content. |
| Detection Method | Fluorescent quantitative PCR (SYBR Green dye-based) |
| Sample Type | cDNA or DNA templates with gene-specific primers; recommended amplicon length 80-300 bp; suitable for templates with 30%-70% GC content. |
| Performance Range / Specifications | Good linearity across a broad quantification range. Recommended annealing temperature: 55-60 °C. Recommended amplicon length: 80-300 bp. Valid amplification efficiency: 90-110% (slope -3.0 to -3.5, R2 > 0.98); reliable Ct range 15-35. |
| Specificity | Formulated with factors that effectively suppress nonspecific PCR amplification; melt curve analysis yields a single peak for specific products. Ligand-based hot start reduces nonspecific priming during reaction setup. |
| Reaction Conditions / Protocol | A two-step protocol is commonly used (combined annealing/extension at 55-60 °C). To increase specificity, use the two-step protocol or raise the annealing temperature; when amplification efficiency is low and Ct values are high, switch to a three-step protocol or extend the extension time. Keep the pre-denaturation step short (about 5 min); prolonged pre-denaturation (e.g., 10 min) can reduce enzyme activity and product yield. |
| Components / Formulation | 2× qPCR SYBR Green Master Mix (High ROX Plus), containing hot-start DNA polymerase, SYBR Green I, dNTPs, Mg2+, and High ROX reference dye Package sizes: 1 mL; 5× 1 mL; 50× 1 mL; 100× 1 mL |
| Storage Conditions | Store at -25 to -15 °C protected from light. Avoid repeated freeze-thaw cycles. The product contains the fluorescent dye SYBR Green I; protect from strong light during storage and reaction setup. |
| Shelf Life | 18 months when stored at -25 to -15 °C protected from light. |
| Package Specifications | 1 mL 5× 1 mL 50× 1 mL 100× 1 mL |
| Product Form | 2× ready-to-use master mix (liquid premix) |
| Concentration | 2× |
| Source / Origin | Recombinant hot-start DNA polymerase with chemically synthesized SYBR Green I dye, dNTPs, Mg2+, and Low ROX reference dye. |
| Expiration Date / Stability | Stable for 18 months at -25 to -15 °C protected from light. Avoid repeated freeze-thaw cycles; protect from strong light during storage and reaction assembly. |
| Compatibility | Compatible with qPCR instruments that require a High ROX reference dye (High ROX instruments). |
| Application Notes / Precautions | A two-step cycling protocol is recommended for most targets. To improve amplification specificity, use a two-step protocol or raise the annealing temperature; if amplification efficiency is low and Ct values are too high, switch to a three-step protocol or extend the extension time. Do not prolong the pre-denaturation step (e.g., from 5 min to 10 min); excessive pre-denaturation can impair enzyme activity and reduce PCR product yield. A valid qPCR result requires: (1) a standard curve with amplification efficiency of 90-110% (slope between -3.0 and -3.5; efficiency = 10^(-1/slope) - 1; a slope of -3.32 corresponds to 100% efficiency) and R2 > 0.98; (2) a sigmoid amplification curve with Ct values between 15 and 35 and a negative control Ct > 35 or undetermined; (3) a single-peak melt curve. Ct values below 15 are discouraged because the fluorescence threshold is set at 10x the standard deviation of the fluorescence signal in cycles 3-15. Minor Tm differences (within 1 degree C) between replicates of the same amplicon are normal and acceptable. If diluting the template unexpectedly lowers the Ct value, inhibitors or impurities in the undiluted template may be suppressing the reaction; dilution can relieve this inhibition. If the reference gene Ct is below 20 while all target genes exceed 30, the targets may be low-abundance transcripts; consider changing the reference gene, redesigning primers, or using a qPCR mix with a wider linear detection range. |
For research use only, not for clinical use.
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