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| Product Name | qPCR SYBR Green Master Mix (ROX Provided Separately) |
| Catalog No. | RESK-0015 |
| Description | This product is a 2× premixed solution for real-time quantitative PCR amplification. The mix contains hot-start DNA polymerase, SYBR Green I, dNTPs, and Mg2+. Only template and primers need to be added to run real-time fluorescent quantitative PCR, greatly simplifying the workflow and reducing the risk of contamination. To accommodate different real-time qPCR instrument models, ROX reference solutions at different concentrations (High ROX/Low ROX) are provided separately for correcting well-to-well fluorescence signal variation. The DNA polymerase ligand used in this product modulates polymerase activity in real time as temperature changes. The formulation includes factors that effectively suppress non-specific PCR amplification and enhance amplification efficiency, enabling excellent linearity over a wide quantitative range. |
| Intended Use | For dye-based (SYBR Green I) real-time fluorescent quantitative PCR. ROX reference dye is provided separately (High ROX/Low ROX), allowing the user to add the appropriate ROX concentration according to the requirements of their specific qPCR instrument. |
| Principle / Technology | SYBR Green I intercalating dye chemistry for real-time fluorescent quantitative PCR, with a hot-start DNA polymerase whose ligand adjusts polymerase activity in response to temperature changes. The formulation contains factors that suppress non-specific amplification and improve amplification efficiency. ROX reference solutions (High ROX/Low ROX) are supplied separately so users can match the ROX level required by their instrument to correct well-to-well fluorescence signal errors. |
| Detection Method | Fluorescent quantitative PCR (SYBR Green dye-based) |
| Sample Type | DNA or cDNA templates. A common input is cDNA reverse-transcribed from 500-1000 ng total RNA, diluted 10-fold, with 1 µL used per qPCR reaction. |
| Performance Range / Specifications | Convenient to use with a simplified workflow: only template and primers need to be added. Good linearity across a wide quantitative range. Recommended amplification length: 80-300 bp. |
| Specificity | The formulation contains factors that effectively suppress non-specific PCR amplification. |
| Reaction Conditions / Protocol | Only template and primers need to be added to the 2× premix; add the separately provided ROX reference solution (High ROX or Low ROX) according to the instrument used. Optimal template input should be determined empirically: dilute the template (generally 5-10 fold) and run a template gradient, selecting the input that gives a Ct value of 20-30. Recommended cycle number: generally 40; up to 45 for low-abundance targets. Recommended amplification length: 80-300 bp. For detailed cycling parameters, refer to the product manual. |
| Components / Formulation | 2× qPCR SYBR Green Master Mix, containing hot-start DNA polymerase, SYBR Green I, dNTPs, and Mg2+ ROX Reference Dye (High ROX), provided separately ROX Reference Dye (Low ROX), provided separately |
| Storage Conditions | Store at -20°C protected from light. Avoid repeated freeze-thaw cycles. The product contains the fluorescent dye SYBR Green I; avoid exposure to strong light during storage and reaction setup. |
| Shelf Life | 18 months. |
| Package Specifications | 1 mL; 5×1 mL; 50×1 mL; 100×1 mL |
| Concentration | 2× |
| Shipping Conditions | Shipped with ice packs. |
| Expiration Date / Stability | Valid for 18 months when stored at -20°C protected from light. Avoid repeated freeze-thaw cycles and exposure to strong light (the mix contains SYBR Green I). |
| Compatibility | Compatible with the vast majority of real-time qPCR instruments. ROX reference solutions (High ROX/Low ROX) are provided separately, allowing users to add the ROX concentration required by their specific instrument model. |
| Application Notes / Precautions | Determine optimal template input empirically: dilute the template DNA (generally 5-10 fold) and test a gradient, choosing the input that yields a Ct of 20-30. A common input is 1 µL of 10-fold-diluted cDNA reverse-transcribed from 500-1000 ng total RNA. Criteria for valid qPCR results: (1) standard curve with amplification efficiency of 90-110% (slope -3 to -3.5) and R²>0.98; (2) S-shaped amplification curve with Ct between 15 and 35, and negative control Ct>35 or no Ct; (3) a single peak in the melt curve. Ct values below 15 are not recommended because the fluorescence threshold is set at 10× the standard deviation of cycles 3-15. ROX is a passive reference dye that normalizes non-PCR-related fluorescence fluctuations and corrects pipetting or well-to-well variation to provide a stable baseline. Jagged amplification curves at plateau may result from low RNA purity (recommended OD260/OD280 = 1.8-2.0, OD260/OD230 > 2.0) or an uncalibrated instrument; increase template dilution, reprepare high-purity RNA, and calibrate the instrument regularly (generally once a year). If the amplification curve fails to reach plateau, possible causes include too little template (Ct around 35), too few cycles, or low reagent amplification efficiency; increase template input, raise cycle number (40, or 45 for low-abundance genes), or verify efficiency with a standard curve. A melt-curve peak with Tm below 80°C usually indicates primer dimers; increase the annealing temperature, raise template input, lower primer concentration, or redesign primers. For miRNA quantification (amplicons of 80-90 bp), peaks at 70-80°C are normal. Double peaks with both Tm values above 80°C suggest poor primer specificity, cross-contamination, or gDNA contamination; check primers by BLAST, work in a clean bench with fresh tips, and include a no-reverse-transcription control. A single but non-sharp peak may indicate non-specific products of similar size; a Tm span within 7°C is acceptable, or confirm a single band by high-resolution agarose gel electrophoresis. |
For research use only, not for clinical use.
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