ROX is an inert fluorescent dye. Some instruments use it to normalise well-to-well fluorescence variation caused by pipetting inconsistency or optical path differences. Applied Biosystems and Thermo Fisher instruments require a ROX passive reference dye, at the instrument-appropriate concentration for each model, for example high or low ROX. Stratagene Mx instruments use low ROX. Roche LightCycler, Bio-Rad CFX, Qiagen Rotor-Gene, and most other platforms use no ROX. Using the wrong ROX level causes noisy baselines and poor replicate agreement.
PCR & qPCR
SYBR Green qPCR Kit and TaqMan Probe-Based Master Mixes for Quantitative PCR
SYBR Green and probe-based qPCR master mixes for accurate gene expression quantification
Dye-based or probe-based chemistry matches your quantification need, target number, and instrument platform. SYBR Green chemistry binds all double-stranded DNA products and is the lower-cost entry point for single-target quantification. Probe-based chemistry uses a sequence-specific hydrolysis probe, adding specificity for multiplexing and for your targets where primer-dimer background is a concern.
SYBR Green master mixes come in No-ROX, Low-ROX, and High-ROX formats, matched to the ROX reference dye requirement of your instrument platform. Fluorescein-compatible formulations are available for instruments that use fluorescein as a passive reference.
For probe-based quantification, standard and direct-amplification formats are available. Standard formats need a clean nucleic acid input. Direct formats are formulated to tolerate common inhibitors in blood, tissue lysates, and swab material, allowing workflows without a separate extraction step for your validated sample types. Standalone probe-based master mixes pair with appropriately dye- and quencher-matched commercially synthesised or custom hydrolysis probes.
Probe selection depends on the target and how many targets you need to detect at once. It also depends on whether your sample needs direct amplification or a clean extract first.
Choose the qPCR chemistry and ROX level for your instrument
1) SYBR Green, single target. A SYBR Green master mix with the ROX level that matches your instrument suits this format. Most ABI/Thermo systems require a ROX-containing mix, with the exact (high or low) level specified in the instrument and kit guidelines. A High-ROX or Low-ROX formulation is recommended for your instrument, Low-ROX for Stratagene Mx, and No-ROX for Roche LightCycler, Bio-Rad CFX, and Qiagen Rotor-Gene. The wrong ROX level causes poor normalisation and noisy baselines.
2) Probe-based, single or multiplex target. A probe-based No-ROX master mix suits most instruments, or a cost-efficient probe 2x master mix works as an alternative. It pairs with a dual-labelled hydrolysis probe from the qPCR Probe and Primer Synthesis section.
3) Direct amplification from unpurified samples. A probe-based direct master mix suits this format. It is formulated to enable amplification from blood, tissue lysates, and swabs for validated protocols, without a separate extraction step.
4) High-throughput or large batch. Large-format packs in 500-reaction sizes give the most economical per-reaction cost for your extended runs. Kits are stable at minus 20 degrees C.
5) Genotyping. For SNP or allelic discrimination assays, a probe-based mix with a high-ROX reference gives the most reliable DeltaDelta-Rn readout. A TaqMan-validated master mix suits your allelic probes.
Compatibility of the chosen master mix, including ROX level and probe dyes, with your specific qPCR instrument and optical setup matters before you order.
Applications
Gene expression profiling
SYBR Green master mixes offer a fast, cost-effective way to profile your defined set of targets, once primer pairs are validated for single-product amplification.
SNP genotyping and allelic discrimination
Probe-based master mixes pair with allele-specific hydrolysis probes to give a reliable DeltaDelta-Rn readout for your genotyping assays.
Viral load measurement
Probe-based chemistry supports quantitative viral load measurement in your clinical research, calibrated against a standard curve of known copy number.
NGS library quantification
qPCR quantifies library concentration before your sequencing runs, giving accurate loading for DNA library preparation workflows.
Multiplexed pathogen panels
Probe-based chemistry detects two or more targets in one tube, supporting your multiplexed pathogen detection panels.
Direct-from-sample testing
Direct-from-sample formats are used in food safety testing and your environmental monitoring, where extraction throughput is a bottleneck.
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Frequently asked questions
SYBR Green I intercalates into double-stranded DNA and emits fluorescence proportional to total amplicon mass. It is easy to use and works with any validated primer pair. However, it cannot distinguish specific product from primer-dimers without a melt-curve step. Probe-based mixes rely on a sequence-specific hydrolysis probe. This probe fluoresces only when it is cleaved during correct amplicon synthesis. That gives higher specificity, enables multiplexing, and removes the need for melt-curve analysis.
Run a standard curve with serial dilutions of a known template, such as a purified PCR product, plasmid, or synthetic gene block. Use at least five log-dilution points, each in triplicate. Calculate amplification efficiency from the slope of the Ct-versus-log-quantity regression (standard curves and efficiency checks are required per MIQE guidelines; Bustin et al. 2009, Clin Chem 55:611). Acceptable efficiency is 90 to 110 percent, which corresponds to a slope of minus 3.1 to minus 3.6. Confirm a single melt-curve peak for SYBR Green assays. Include a no-template control and, for RT-qPCR, a no-reverse-transcriptase control in every plate.
Yes. Absolute quantification needs a standard curve prepared from a material with a known copy number, such as a linearised plasmid or synthetic oligonucleotide. This applies when the assay is calibrated with appropriate standards and run within the validated dynamic range. Unknown sample Ct values are then interpolated against the standard curve to get copy numbers per reaction or per input mass. This approach is used for viral load assays, GMO quantification, and copy-number variation studies. Accurate results depend on the standard curve and samples sharing the same amplification efficiency.
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