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| Product Name | qPCR SYBR Green Master Mix with Pipetting Tracking Dye (Low ROX) |
| Catalog No. | RESK-0004 |
| Description | This 2× real-time quantitative PCR master mix combines high fluorescence intensity, high sensitivity, strong specificity, and high amplification yield. The core hot-start Taq DNA polymerase is blocked by a specific antibody, effectively suppressing non-specific amplification caused by primer annealing during reaction setup. The formulation includes factors that enhance PCR amplification efficiency and balance the amplification of genes with different GC contents (30–70%), delivering excellent linearity across a wide quantification range. A built-in color-change tracking system made from mixed indicator dyes makes pipetting steps clearly visible, effectively reducing the risk of missed or incorrect sample loading. |
| Intended Use | Dye-based real-time quantitative PCR (qPCR) for gene expression analysis and nucleic acid quantification on Low ROX instruments. |
| Principle / Technology | Antibody-based hot-start Taq DNA polymerase combined with SYBR Green dye-based detection; a color-changing pipetting tracking system (mixed indicator dyes) allows visual monitoring of sample loading to minimize pipetting errors. |
| Detection Method | Fluorescent quantitative PCR (SYBR Green dye-based) |
| Sample Type | cDNA and DNA templates from various sample types (validated on mouse, human cell line, and Arabidopsis targets). |
| Performance Range / Specifications | Color-change pipetting tracking reduces the risk of missed or incorrect loading. Excellent linearity across a wide dynamic range (up to 7 orders of magnitude of template). Precisely distinguishes 2-fold differences in template concentration. Small well-to-well variation for superior reproducibility. Assembled reactions remain stable at room temperature for 24 hours; performance is unaffected by 20 freeze-thaw cycles. Compatible with both standard and fast cycling programs (denaturation 10 s/extension 30 s or 3 s/10 s). |
| Sensitivity / LOD | Reliably detects templates across a 10^1–10^7 copy gradient and precisely distinguishes 2-fold template concentration differences. |
| Specificity | Antibody-mediated hot start effectively suppresses non-specific amplification arising from primer annealing during reaction setup; formulation factors further inhibit non-specific PCR amplification. |
| Reaction Conditions / Protocol | Compatible with standard programs (denaturation 10 s, extension 30 s) and fast programs (denaturation 3 s, extension 10 s). Mix by gentle inversion followed by brief centrifugation; do not vortex vigorously. Recommended amplicon length: 80–300 bp. |
| Components / Formulation | qPCR SYBR Green Master Mix with Pipetting Tracking Dye (Low ROX), 2×: 1 mL / 5× 1 mL / 100× 1 mL 10× Dilution Buffer: 1 mL / 1 mL / 20× 1 mL |
| Storage Conditions | Store at -25 to -15°C, protected from light. |
| Shelf Life | 18 months when stored at -25 to -15°C, protected from light. |
| Package Specifications | 1 mL 5× 1 mL |
| Product Form | Premixed solution (master mix) |
| Quality Control | Assembled reactions show ≤0.5 CT difference after 24 h at room temperature (protected from light), after 14 days at 25°C or 7 days at 37°C, and after 20 freeze-thaw cycles. |
| Concentration | 2× |
| Expiration Date / Stability | Stable for 18 months at -25 to -15°C protected from light; reagent retains performance after 14 days at 25°C, 7 days at 37°C, or 20 freeze-thaw cycles (CT variation ≤0.5). |
| Compatibility | Compatible with qPCR instruments requiring Low ROX reference dye. Recommended reaction volume is 20 μL; halving the reaction volume may lower the total ROX concentration and trigger BadRox errors on some instruments. |
| Application Notes / Precautions | Mix the master mix by gentle inversion and brief centrifugation; avoid vigorous shaking. The 10× Dilution Buffer is optional; if used, ensure it is at 1× final concentration after mixing with the template. cDNA template can be used undiluted or diluted; aim for target CT values between 15 and 30 (each 10-fold dilution increases CT by ~3.3). Assembled reactions can be kept at 4°C or room temperature for up to 24 h if protected from light and not left open; gently mix and spin before loading. Primer design: amplicon 80–300 bp; primer length 17–25 bases; GC content 40–60%; Tm difference between forward and reverse primers ≤1°C; avoid long complementary stretches; verify specificity by BLAST and design across introns where possible. If no amplification curve appears, check that signal acquisition is enabled (at the annealing/extension step for two-step protocols), confirm amplicon length is below 500 bp, and verify primer and template integrity. For NTC amplification, analyze the melt curve to distinguish primer-dimers from contamination; a ΔCT ≥5 between NTC and target indicates negligible impact. CT values in the NRT control indicate genomic DNA contamination; use RNA extraction with genomic DNA removal or design primers across introns. qPCR products can be analyzed by gel electrophoresis, but loading buffer must be added before loading. |
For research use only, not for clinical use.
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