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| Product Name | qPCR SYBR Green Master Mix with Pipetting Tracking Dye (High ROX) |
| Catalog No. | RESK-0008 |
| Description | qPCR SYBR Green Master Mix with Pipetting Tracking Dye (High ROX) is a 2× premix for real-time quantitative PCR with high fluorescence intensity, high sensitivity, strong specificity, and high amplification yield. Its core component is an antibody-based hot-start Taq DNA polymerase that effectively suppresses nonspecific amplification caused by primer annealing during sample preparation. The formulation contains factors that boost amplification efficiency and balance amplification of genes with different GC contents (30%-70%), giving excellent linearity across a broad quantification range. A color-change reaction between blended dyes monitors pipetting during setup, clearly showing sample addition progress and effectively reducing the risk of missed or incorrect additions. |
| Intended Use | Dye-based real-time quantitative PCR on High ROX instruments, with built-in color-change pipetting tracking to reduce liquid-handling errors; suitable for standard and fast cycling programs. For research use. |
| Principle / Technology | SYBR Green I dye-based fluorescent detection with an antibody hot-start Taq DNA polymerase that remains inactive during reaction setup and is activated by heat, minimizing nonspecific priming and primer-dimer formation. Buffer enhancers improve amplification efficiency and equalize amplification across 30%-70% GC content. Pipetting tracking is achieved through a color-change reaction between different dyes: mixing the colored master mix with template produces a visible color shift, allowing visual confirmation that each well has been correctly loaded. |
| Detection Method | Fluorescent quantitative PCR (SYBR Green dye-based) |
| Sample Type | DNA or cDNA templates from various sample types (validated with mouse, human 293 cell, and Arabidopsis targets) with gene-specific primers; recommended amplicon length 80-300 bp. |
| Performance Range / Specifications | Reliable detection across 7 orders of magnitude (10^1-10^7 copies of plasmid template) with good linearity over a broad dynamic range. Accurately discriminates 2-fold differences in template concentration. Small well-to-well variation between replicates for superior reproducibility. Compatible with standard programs (10 s denaturation, 30 s extension) and fast programs (3 s denaturation, 10 s extension), with strongly correlated Ct values between the two. Assembled reactions remain stable at room temperature for 24 hours (ΔCt within 0.5); reagent stable after 14 days at 25 °C or 7 days at 37 °C, and after 20 freeze-thaw cycles (ΔCt within 0.5). |
| Sensitivity / LOD | Reliably detects templates down to 10^1 copies within a validated 10^1-10^7 copy dynamic range. |
| Specificity | Antibody-based hot start effectively suppresses nonspecific amplification from primer annealing during setup; outperforms other qPCR reagents across multiple sample types and gene targets, as shown by lower ΔCt values. |
| Reaction Conditions / Protocol | Suitable for standard or fast programs. Standard program: denaturation 10 s, extension 30 s per cycle. Fast program: denaturation 3 s, extension 10 s per cycle. Mix the reagent by gentle inversion followed by brief centrifugation before use; do not vortex vigorously. An optional 10× Dilution Buffer (yellow) is provided for template dilution: e.g., dilute 20 µL cDNA with 180 µL sterile ultrapure water (10-fold), then mix 90 µL diluted template with 10 µL 10× Dilution Buffer so the buffer is at 1× in the final reaction; its use is optional and does not affect performance. Assembled reactions can be held protected from light at 4 °C or room temperature for up to 24 hours; keep reactions covered, mix gently and centrifuge before loading, and check that tubes/plates are clean. |
| Components / Formulation | 2× qPCR SYBR Green Master Mix (High ROX) with pipetting tracking dye, containing antibody hot-start Taq DNA polymerase, SYBR Green I, dNTPs, Mg2+, and High ROX reference dye 10× Dilution Buffer (yellow) Package sizes: 1 mL; 5× 1 mL |
| Storage Conditions | Store at -25 to -15 °C protected from light. |
| Shelf Life | 1 year when stored at -25 to -15 °C protected from light. |
| Package Specifications | 1 mL 5× 1 mL |
| Product Form | 2× ready-to-use liquid premix with color-change pipetting tracking dye |
| Concentration | 2× |
| Source / Origin | Antibody hot-start Taq DNA polymerase with optimized buffer, SYBR Green I, tracking dyes, and High ROX reference dye. |
| Expiration Date / Stability | Shelf life 1 year at -25 to -15 °C protected from light. The reagent retains performance after 14 days at 25 °C or 7 days at 37 °C, and after 20 freeze-thaw cycles (Ct differences within 0.5). Assembled reaction mixes remain stable for 24 hours at room temperature when protected from light (ΔCt within 0.5). |
| Compatibility | Compatible with qPCR instruments requiring a High ROX reference dye (High ROX instruments). Use a 20 µL reaction volume; half-volume (10 µL) reactions lower the total ROX concentration and increase the risk of BadRox errors. |
| Application Notes / Precautions | Mix before use by gentle inversion and brief centrifugation; do not vortex vigorously. Template dilution: use undiluted or diluted cDNA so that target Ct values fall between 15 and 30; each 10-fold dilution of cDNA increases Ct by approximately 3.3. The provided 10× Dilution Buffer is optional. If the assembled reaction cannot be run immediately, keep it covered and protected from light at 4 °C or room temperature for up to 24 hours; mix gently and centrifuge before loading. Primer design guidelines: amplicon 80-300 bp; primer length 17-25 bases; GC content 40-60% (45-55% optimal); forward and reverse Tm within 1 °C; avoid runs of T/C or A/G; 3' end preferably G or C, avoid terminal T and GC- or AT-rich 3' ends; avoid complementary stretches of 3 or more bases within or between primers (2 or more at 3' ends); confirm specificity by BLAST and design across introns where possible. BadRox errors usually result from an uncalibrated or aging instrument (annual calibration recommended) or from using 10 µL reactions; use the standard 20 µL volume. qPCR products can be checked by melt curve analysis; if gel electrophoresis is required, add loading buffer before loading—products cannot be loaded directly. No amplification curve: confirm signal collection is enabled (set to the annealing/extension step for two-step protocols); keep amplicons at 80-300 bp; check primer design and integrity (PAGE if degradation is suspected); re-prepare template if degraded. NTC amplification: judge by melt curve. A non-overlapping peak with lower Tm indicates primer-dimers and does not affect gene signal if the gene peak is single; an overlapping peak indicates contamination—check water, primers, and reagents, and consider aerosol contamination; a ΔCt (NTC vs gene) of 5 or more indicates negligible impact. Ct in the no-RT control indicates genomic DNA contamination; use RNA extraction with genomic DNA removal, add a gDNA digestion step in reverse transcription, or design primers across introns. Poor replicate agreement: for Ct < 30, improve pipetting accuracy (prepare bulk mixes, avoid small-volume pipetting, calibrate pipettes annually); for Ct >= 30, low template abundance increases stochastic variation—add 2-3 extra replicates and exclude outliers during analysis. Extra melt-curve peaks: a peak left of the main peak suggests primer-dimers (raise annealing temperature, lower primer concentration, or redesign primers); a peak right of the main peak suggests nonspecific amplification from poor primer specificity (redesign primers) or genomic DNA contamination (re-prepare cDNA with gDNA removal). |
For research use only, not for clinical use.
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