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| Product Name | qPCR SYBR Green Master Mix with Pipetting Tracking Dye (No ROX) |
| Catalog No. | RESK-0002 |
| Description | This 2× ready-to-use premix for real-time quantitative PCR delivers high fluorescence intensity, excellent sensitivity, strong specificity, and high amplification yield. Its core component, an antibody-mediated hot-start Taq DNA polymerase, effectively suppresses non-specific amplification caused by primer annealing during reaction setup. The formulation also incorporates factors that boost PCR amplification efficiency and balance the amplification of genes with different GC contents (30-70%), enabling reliable linearity across a wide quantitative range. A color-change dye system built on the mixing of differently colored dyes makes every pipetting step visible, effectively reducing the risk of missed or incorrect additions during reaction setup. |
| Intended Use | Dye-based real-time quantitative PCR (qPCR) for gene expression analysis and nucleic acid quantification on qPCR instruments that do not require ROX reference dye. |
| Principle / Technology | Antibody-mediated hot-start Taq DNA polymerase with SYBR Green I dye chemistry; amplification-enhancing and GC-balancing factors support targets with 30-70% GC content, while a mixed-dye color-change system provides visual pipetting tracking. |
| Detection Method | Fluorescent quantitative PCR (SYBR Green dye-based) |
| Sample Type | cDNA and DNA templates (e.g., plasmid DNA) |
| Performance Range / Specifications | Reliably detects template amounts across 7 orders of magnitude (10^1-10^7 plasmid copies) with excellent linearity over a wide dynamic range. Precisely distinguishes 2-fold differences in template concentration. Minimal well-to-well variation between replicates, delivering superior reproducibility. Prepared reaction mixes remain stable at room temperature for 24 hours, and the product withstands 20 freeze-thaw cycles without performance loss. Compatible with both standard and fast qPCR cycling programs, with strongly correlated Ct values between the two. |
| Sensitivity / LOD | High sensitivity with reliable detection across a 7-log dynamic range down to low-copy templates; strong fluorescence signal supports accurate quantification. |
| Specificity | Antibody-mediated hot-start Taq DNA polymerase blocks enzyme activity during reaction setup, effectively suppressing non-specific amplification arising from premature primer annealing. |
| Reaction Conditions / Protocol | Standard program: denaturation 10 s, annealing/extension 30 s per cycle. Fast program: denaturation 3 s, annealing/extension 10 s per cycle. Ct values obtained with standard and fast programs are strongly correlated. |
| Components / Formulation | 2× qPCR SYBR Green Master Mix (No ROX): 1 mL / 5 × 1 mL / 100 × 1 mL, depending on package size 10× Dilution Buffer: 1 mL / 1 mL / 20 × 1 mL, depending on package size |
| Storage Conditions | Store at -25°C to -15°C, protected from light |
| Shelf Life | 18 months |
| Package Specifications | 1 mL; 5 × 1 mL |
| Product Form | 2× premix |
| Concentration | 2× |
| Expiration Date / Stability | Maintains performance after storage at 25°C for 14 days or 37°C for 7 days, with Ct differences of less than 0.5. Stable through 20 freeze-thaw cycles, with Ct differences of less than 0.5. Prepared reaction mixes can be kept protected from light at room temperature for 24 hours with no more than 0.5 Ct variation. |
| Compatibility | Designed for No ROX qPCR instruments; compatible with both standard and fast qPCR cycling programs. |
| Application Notes / Precautions | Mix the reagent by gentle inversion followed by a brief spin; do not vortex vigorously. The 10× Dilution Buffer is optional; if used, ensure a final 1× concentration after mixing with the template (e.g., dilute 20 μL cDNA with 180 μL sterile water, then add 90 μL diluted template to 10 μL 10× Dilution Buffer). cDNA may be used undiluted or diluted; target Ct values between 15 and 30. A 10-fold cDNA dilution typically raises Ct by about 3.3. Prepared reactions can be stored protected from light at 4°C or room temperature for up to 24 hours; mix gently and spin before loading, and never leave the reaction system uncovered. Primer design: amplicon 80-300 bp; primer length 17-25 nt; GC content 40-60% (45-55% optimal); forward and reverse Tm within 1°C; avoid long complementary stretches and confirm specificity by BLAST; design across introns where possible. For 'BadRox' instrument errors: calibrate the instrument regularly and use a 20 μL reaction volume rather than 10 μL. qPCR products may be analyzed by gel electrophoresis, but loading buffer is required; specificity is usually assessed by melt curve analysis. If no amplification curve appears, confirm signal acquisition is enabled (set at the annealing/extension step for two-step protocols), keep amplicons at 80-300 bp, verify primer integrity, and rule out template degradation. Ct in NTC wells: analyze the melt curve - a lower-Tm, non-overlapping peak indicates primer-dimers (usually no impact); an overlapping peak indicates contamination, and a ΔCt ≥ 5 versus the target generally means the impact is negligible. Ct in NRT wells indicates genomic DNA contamination; use RNA extraction with gDNA removal or add a gDNA digestion step during reverse transcription, or design intron-spanning primers. Poor replicate reproducibility: improve pipetting accuracy with larger master mixes and calibrated pipettes; for low-abundance templates (Ct ≥ 30), add 2-3 extra replicates and exclude outliers. Extra peaks in the melt curve: peaks left of the main peak suggest primer-dimers (raise annealing temperature, lower primer concentration, or redesign primers); peaks to the right suggest non-specific amplification or gDNA contamination. |
For research use only, not for clinical use.
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