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| Product Name | 5′-ROX (Carboxy-X-Rhodamine) Labeled Oligonucleotide |
| Catalog No. | PRIPRO-0008 |
| Description | A custom DNA oligonucleotide with 5′-terminal carboxy-X-rhodamine (ROX) fluorescent labeling. ROX is a red fluorophore with excitation maximum at approximately 585 nm and emission at 605 nm, well separated from the green and yellow fluorophores (FAM, HEX, TET). This spectral position makes ROX the reference dye of choice in many qPCR master mixes for well-to-well normalization (passive reference). It also serves as a detection label for multiplex qPCR and fragment analysis. The dye is conjugated via a phosphoramidite linker during synthesis and the product is HPLC purified to remove free dye, which can cause high background fluorescence. |
| Intended Use | Designed as a passive reference dye for qPCR normalization, as a fluorescent label for multiplex PCR detection in the ROX channel, for fragment analysis and SNP genotyping on capillary sequencers, and as a FRET acceptor for FAM and HEX donors. |
| Principle / Technology | 5′-ROX phosphoramidite coupling in solid-phase synthesis; HPLC purification to isolate dye-labeled full-length product |
| Detection Method | UV-Vis (A260, A585); fluorescence spectroscopy (Ex 585 nm / Em 605 nm); HPLC with fluorescence and UV dual detection |
| Sample Type | PCR reactions for normalization; multiplex genotyping panels; FRET assay samples |
| Performance Range / Specifications | 10–60 bases; ROX labeling confirmed by A585/A260 ratio |
| Sensitivity / LOD | ROX fluorescence detectable at 0.5–2 nM on qPCR platforms; passive reference signal stable across 40+ cycles |
| Specificity | Minimal spectral overlap with FAM, HEX, and TET; ideal for four-color multiplex panels |
| Reaction Conditions / Protocol | Resuspend at 100 µM in nuclease-free water or TE buffer. For passive reference, use at 30–100 nM final in qPCR reactions. Protect from light throughout. |
| Components / Formulation | Light-protected tube with lyophilized ROX-labeled oligo |
| Storage Conditions | –20 °C in the dark; use amber tubes and avoid light exposure |
| Shelf Life | 12 months at –20 °C dry (dark); 6 months in solution at –20 °C (dark) |
| Package Specifications | Foil-wrapped screw-cap tube; available at 5, 10, 25, 50 nmol scale |
| Product Form | Lyophilized pink to magenta pellet |
| Quality Control | HPLC purity ≥90%; MALDI-TOF MS; A585 and A260 quantification; dye incorporation efficiency >85%; residual free dye <5% by HPLC; fluorescence emission scan. |
| Key Features | Standard passive reference dye for qPCR normalization; well-separated red fluorescence channel; low background when free dye removed by HPLC; compatible with all ROX-requiring master mixes; FRET acceptor for green/yellow donors. |
| Purity | ≥90% by HPLC; residual free dye <5% |
| Concentration | Resuspend to 100 µM stock; actual yield quantified by A260 measurement corrected for dye absorbance contribution |
| Activity / Unit Definition | Extinction coefficient ~82,000 M⁻¹cm⁻¹ at 585 nm; emission maximum 605 nm; quantum yield ~0.5 |
| Molecular Weight | Oligonucleotide MW plus approximately 630 Da for ROX linker moiety |
| Source / Origin | Synthetic oligonucleotide with chemically conjugated ROX fluorophore |
| pH Range / Optimal pH | pH-insensitive fluorescence in range 5.0–9.0; robust for qPCR buffer conditions |
| Shipping Conditions | Ambient temperature in light-protective packaging |
| Expiration Date / Stability | 12 months dry at –20 °C (dark); 6 months in solution at –20 °C (dark) |
| Regulatory / Compliance | For research use only; RUO grade |
| Compatibility | ROX channel on ABI 7500/7900HT, QuantStudio series, Bio-Rad CFX series, Agilent Mx3000P/Mx3005P; passive reference dye for TaqMan Universal Master Mix and SYBR Green Master Mix (ROX-requiring formulations) |
| Recommended Buffer System | Nuclease-free water or TE buffer pH 8.0; ROX fluorescence is pH-stable |
| Application Notes / Precautions | Check manufacturer instructions for optimal ROX concentration in your specific qPCR master mix (typically 30–100 nM final). Pre-dilute in master mix rather than adding separately to each well for better well-to-well uniformity. |
| Batch-to-Batch Consistency | A585/A260 ratio consistent within ±10%; fluorescence intensity per OD unit maintained across production lots |
For research use only, not for clinical use.
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