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| Product Name | Molecular Beacon Probe Custom Synthesis Service |
| Catalog No. | PRIPRO-0015 |
| Description | A custom molecular beacon synthesis service producing stem-loop structured oligonucleotide probes for real-time PCR, in situ hybridization, and live-cell RNA imaging. Molecular beacons are single-stranded oligonucleotides forming a hairpin structure with a fluorophore at the 5′ end, a quencher at the 3′ end, a loop region complementary to the target, and a short self-complementary stem (typically 5–7 bases) that holds the fluorophore and quencher in close proximity when the beacon is unbound. Upon target hybridization, the stem opens, separating fluorophore from quencher and generating a bright fluorescent signal. Each beacon is custom-designed for the user's target sequence, with design optimization performed by experienced scientists. The service includes HPLC purification and rigorous quality control to ensure proper folding, high signal-to-background ratio, and sequence specificity. |
| Intended Use | Designed for real-time PCR with molecular beacon detection chemistry, live-cell mRNA imaging and tracking, in situ hybridization (FISH), SNP and mutation detection with single-base discrimination, and multiplex gene expression analysis in single cells. |
| Principle / Technology | Hairpin stem-loop structure: 5′ fluorophore and 3′ quencher held together by stem hybridization; target binding opens stem, separating dye from quencher to produce fluorescence; higher specificity than linear probes due to conformational constraint |
| Detection Method | Fluorescence dequenching upon target hybridization; signal measured in real-time or endpoint format; single-base mismatch discrimination verified during QC |
| Sample Type | Purified nucleic acids for qPCR; fixed or live cells for in situ imaging; tissue sections for FISH |
| Performance Range / Specifications | Loop 15–25 bases; stem 5–7 bases; total length 25–40 bases; melting temperature of stem 5–7 °C above assay temperature; loop Tm optimized for target binding at assay temperature |
| Sensitivity / LOD | Single-molecule detection achievable in live-cell imaging under optimized conditions; qPCR detection limit comparable to TaqMan probes (<10 copies per reaction) |
| Specificity | Single-base discrimination capability; stem-loop structure provides thermodynamic barrier to mismatched target binding; higher specificity than linear probes for SNP detection |
| Reaction Conditions / Protocol | Use at 100–300 nM in qPCR. For live-cell imaging, microinject or transfect at 0.5–2 µM final. Include 2–5 mM Mg²⁺ for stem stability. Pre-anneal by heating to 95 °C and slow-cooling before first use. |
| Components / Formulation | Custom-designed dual-labeled molecular beacon; design documentation with folding prediction; certificate of analysis |
| Storage Conditions | –20 °C in dark; protect from light; aliquot to avoid freeze-thaw |
| Shelf Life | 12 months at –20 °C dry (dark); 6 months at –20 °C in solution (dark) |
| Package Specifications | Light-protective tube; scale options 5–100 nmol; design report included |
| Product Form | Lyophilized dual-modified oligonucleotide (hairpin-capable); color by dye |
| Quality Control | HPLC ≥95% dual-labeled; MALDI-TOF MS; hairpin folding verified by thermal denaturation profile; signal-to-background ratio >10:1 confirmed with complementary target; residual free dye <2%. |
| Key Features | Single-base mismatch discrimination; higher specificity than linear probes; self-reporting — no need for probe cleavage; suitable for live-cell applications; custom design with expert support; wide fluorophore-quencher pair selection. |
| Purity | ≥95% dual-labeled (HPLC); correct folding confirmed by melt curve analysis |
| Concentration | Resuspend to 100 µM in nuclease-free water; actual concentration reported on COA with A260 measurement |
| Activity / Unit Definition | Signal-to-background ratio >10:1 with fully complementary target; signal-to-noise >20:1 for optimized designs; negligible signal with single-mismatch target |
| Molecular Weight | Sequence-dependent; fluorophore and quencher contributions reported on COA; verified by MS |
| Source / Origin | Synthetic; all modifications from chemical synthesis |
| pH Range / Optimal pH | Functional pH 6.5–8.5; optimal folding at pH 7.5–8.0 with Mg²⁺ present |
| Shipping Conditions | Ambient temperature, light-protected; lyophilized beacons stable during transit |
| Expiration Date / Stability | 12 months dry at –20 °C (dark); 6 months at –20 °C in TE with MgCl₂ (dark); avoid repeated freeze-thaw |
| Regulatory / Compliance | For research use only; not for diagnostic applications |
| Compatibility | Compatible with all qPCR platforms having appropriate fluorophore channels; suitable for fluorescence microscopy with standard filter cubes; applicable to flow cytometry for cell-based assays |
| Recommended Buffer System | Tris-HCl pH 7.5–8.0 with 2–5 mM MgCl₂ for stem stability; avoid EDTA in working buffer (chelates Mg²⁺ and destabilizes stem) |
| Application Notes / Precautions | Pre-anneal molecular beacon before first use: heat to 95 °C for 2 min, then cool slowly to room temperature over 30 min. For live-cell applications, use nuclease-resistant backbone modifications (phosphorothioate or 2′-O-Me) to prevent degradation. |
| Batch-to-Batch Consistency | HPLC purity ≥95%; signal-to-background ratio within ±15%; melting temperature (stem Tm) within ±1 °C across lots |
For research use only, not for clinical use.
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