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| Product Name | Dual-Labeled Hydrolysis Probe (ROX-BHQ2) for Multiplex qPCR |
| Catalog No. | PRIPRO-0012 |
| Description | A dual-labeled qPCR hydrolysis probe with 5′-ROX (carboxy-X-rhodamine) reporter and 3′-Black Hole Quencher 2 (BHQ2) dark quencher. ROX emits in the red spectral region (Ex 585 nm / Em 605 nm), providing excellent separation from FAM, HEX, and TET channels for high-order multiplex (three or four colors). BHQ2 is optimized for quenching longer-wavelength fluorophores (550–650 nm range), making it the ideal partner for ROX. The probe functions via TaqMan chemistry: intact probe fluorescence is suppressed by combined FRET and static quenching; Taq polymerase exonuclease cleavage during PCR separates ROX from BHQ2, generating a red fluorescence signal. HPLC purification ensures >95% dual-labeled homogeneity, which is critical for low background and high signal-to-noise ratio in multiplex panels. |
| Intended Use | Designated for three- or four-color multiplex qPCR assays, including multi-pathogen respiratory panels, multi-gene expression profiling, internal positive control detection alongside target genes, and allellic discrimination with multi-probe single-well reactions. |
| Principle / Technology | FRET and static quenching of ROX by BHQ2; probe cleavage by Taq polymerase exonuclease activity liberates ROX for real-time fluorescence detection in red channel |
| Detection Method | Real-time fluorescence acquisition in ROX channel (Ex 585 nm / Em 605 nm); simultaneous multi-channel monitoring in multiplex format |
| Sample Type | Purified nucleic acids; multiplex panels accommodating up to four probes per well (FAM, HEX, ROX, CY5 channels) |
| Performance Range / Specifications | Probe 18–30 bases; Tm 65–70 °C; GC 40–60%; spectral separation >50 nm from FAM and >30 nm from HEX emission maxima |
| Sensitivity / LOD | <10 copies per reaction detected in multiplex format; dynamic range of 6–7 orders of magnitude maintained in multicolor panels |
| Specificity | Sequence-specific probe hybridization and cleavage; BHQ2 absorption spectrum (550–650 nm) matched to ROX emission; negligible quenching of shorter-wavelength fluorophores |
| Reaction Conditions / Protocol | Use at 100–250 nM in multiplex qPCR. For four-color panels, adjust each probe concentration to balance endpoint fluorescence. Standard two-step cycling with multi-channel acquisition. |
| Components / Formulation | Light-protected tube with lyophilized ROX-BHQ2 probe; COA included |
| Storage Conditions | –20 °C in dark container; protect from light at all times |
| Shelf Life | 12 months at –20 °C dry (dark); 6 months in solution at –20 °C (dark) |
| Package Specifications | Foil-wrapped amber tube; 5–50 nmol scale; COA with HPLC and MS data |
| Product Form | Lyophilized pink to red pellet |
| Quality Control | HPLC purity ≥95% dual-labeled; MALDI-TOF MS; quenching efficiency >95% confirmed; spectral purity verified by fluorescence scan; residual free dye <3%. |
| Key Features | Red channel detection for multiplex qPCR panels; BHQ2 dark quencher optimized for long-wavelength fluorophores; >95% HPLC purity; mass spectrometry confirmation; compatible with ROX channel on all major qPCR instruments. |
| Purity | ≥95% dual-labeled by HPLC; free dye <3%; single-labeled <2% |
| Concentration | Lyophilized; resuspend to 100 µM based on A260 plus ROX absorbance correction; typical 15–40 OD260 at 25 nmol scale |
| Activity / Unit Definition | Quenching efficiency >95% for ROX-BHQ2 pair; >8-fold fluorescence increase upon enzymatic cleavage; BHQ2 extinction coefficient >60,000 M⁻¹cm⁻¹ at ~600 nm |
| Molecular Weight | Probe sequence MW plus ~630 Da (ROX) and ~560 Da (BHQ2); COA provides accurate mass |
| Source / Origin | Synthetic; ROX and BHQ2 chemical building blocks |
| pH Range / Optimal pH | ROX fluorescence pH-independent 5.0–9.0; probe stable in qPCR buffer pH 8.0–8.5 |
| Shipping Conditions | Ambient temperature in light-protective packaging |
| Expiration Date / Stability | 12 months dry at –20 °C (dark); 6 months in TE or water at –20 °C (dark); minimize freeze-thaw cycles |
| Regulatory / Compliance | For research use only; not for diagnostic procedures |
| Compatibility | ROX channel on ABI 7500/7900HT/QuantStudio, Bio-Rad CFX series, Roche LightCycler 480, Agilent AriaMx; compatible with commercial multiplex qPCR master mixes |
| Recommended Buffer System | Commercial 2X qPCR master mixes; Tris-based pH 8.0–8.5 with KCl and MgCl₂ 3–5 mM |
| Application Notes / Precautions | In four-color multiplex, test individual probes in single-color reactions first to validate linearity and sensitivity. Perform color compensation using pure dye matrix standards. Balance multiplex probe concentrations for uniform endpoint signals. |
| Batch-to-Batch Consistency | HPLC dual-label purity maintained ≥95%; quenching efficiency within ±3%; fluorescence emission peak within ±3 nm across lots |
For research use only, not for clinical use.
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