The Universal SYBR qPCR Master Mix Passive ROX is a highly optimized reagent designed for real-time quantitative PCR (qPCR) applications. It enables high sensitivity and specificity, making it ideal for gene expression analysis, pathogen detection, and molecular diagnostics. The inclusion of Passive ROX as a reference dye ensures compatibility with a wide range of qPCR instruments.
Key Features and Benefits
- High Sensitivity and Specificity: Detects low-abundance targets with minimal background.
- Optimized for qPCR Applications: Works efficiently across various qPCR platforms.
- Passive ROX Normalization: Reduces signal variation for consistent and accurate results.
- Stable and Robust Formulation: Provides reliable amplification under diverse conditions.
- Ready-to-Use Format: Simplifies workflow and minimizes pipetting errors.
- Compatible with SYBR Green Detection: Enables fluorescent signal generation for quantification.
- Wide Dynamic Range: Ensures accurate quantification across various template concentrations.
- Rapid and Efficient Amplification: Supports high-speed qPCR workflows for improved throughput.
Mechanism of Action
SYBR Green I Dye intercalates with double-stranded DNA (dsDNA) during amplification, producing a fluorescence signal proportional to the DNA concentration. Passive ROX serves as an internal control to correct for instrument-related signal variations, ensuring reproducible and accurate qPCR results. The robust formulation also minimizes non-specific amplification and primer-dimer formation, improving overall qPCR performance.
Applications
- Gene Expression Analysis: Detects differential gene expression with high precision and sensitivity.
- Pathogen Detection: Identifies bacterial and viral DNA in clinical, environmental, and food samples.
- Genotyping Studies: Differentiates genetic variants with high specificity and accuracy.
- MicroRNA and lncRNA Analysis: Quantifies small RNA molecules, enabling research in non-coding RNA functions.
- DNA Quantification for NGS: Measures DNA input for next-generation sequencing (NGS) library preparation.
- Biomarker Discovery: Facilitates identification of disease-associated genetic signatures.
- Forensic and Agricultural Testing: Ensures accurate DNA detection for forensic investigations and crop improvement.
Performance Specifications
| Feature | Universal SYBR qPCR Master Mix Passive ROX |
|---|---|
| Detection Method | SYBR Green fluorescence |
| Passive Reference Dye | ROX |
| Sensitivity | High (detects low copy numbers) |
| Instrument Compatibility | Multiple qPCR platforms |
| Reaction Stability | Long-term storage stability |
| Efficiency | > 90% for most qPCR assays |
| Specificity | Low primer-dimer formation |
Storage and Stability
- Store at -20°C for long-term use.
- Avoid repeated freeze-thaw cycles to maintain reagent integrity.
- Ready-to-use mix ensures stable performance over time.
- Store in light-protected conditions to prevent degradation of fluorescent dye components.
Safety Considerations
- Handle with gloves and protective eyewear to prevent contamination.
- Dispose of waste according to laboratory biosafety guidelines.
- Follow manufacturer-recommended handling procedures to ensure user safety.
- Work in a clean, RNAse-free environment to avoid contamination of qPCR reactions.
Protocol Guidelines
- Reaction Setup:
- Prepare qPCR reactions by mixing Universal SYBR qPCR Master Mix, primers, template DNA, and water.
- Use an optimized primer concentration to enhance specificity.
- Maintain a consistent reaction volume across samples for accuracy.
- Thermal Cycling:
- Follow instrument-specific cycling conditions for denaturation, annealing, and extension.
- Use an optimized annealing temperature to maximize amplification efficiency.
- Fluorescence Detection:
- Monitor SYBR Green fluorescence during the extension phase.
- Passive ROX ensures correction of well-to-well variations for reliable quantification.
- Data Analysis:
- Normalize data using Passive ROX for accurate quantification.
- Perform melt curve analysis to verify the specificity of amplification.
- Use standard curves for absolute quantification of target genes.
- Validation:
- Include no-template controls (NTCs) to check for contamination.
- Perform technical replicates for enhanced data reliability.
Troubleshooting Guide
| Issue | Possible Cause | Solution |
| No amplification | Incorrect reagent setup | Verify reagent volumes and template quality |
| High background signal | Primer-dimer formation | Optimize primer design and concentration |
| Low efficiency | Poor template quality | Use high-quality purified DNA or cDNA |
| Inconsistent results | Pipetting errors | Use calibrated pipettes and proper mixing |
Conclusion
The Universal SYBR qPCR Master Mix Passive ROX is a versatile, high-performance reagent that enhances the accuracy and reproducibility of qPCR experiments. Its stable formulation, Passive ROX normalization, broad instrument compatibility, and superior sensitivity make it an essential tool for molecular biology, diagnostics, and genetic research.
With its advanced formulation ensuring high specificity, efficiency, and low background, this reagent is ideal for researchers looking for reliable and consistent qPCR results across diverse applications, from biomedical research to clinical diagnostics and environmental monitoring.
For standardized protocols and additional details, visit authoritative sources such as the National Institutes of Health (NIH), Centers for Disease Control and Prevention (CDC), and National Science Foundation (NSF).
