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| Product Name | Cy5-Labeled Oligonucleotide Probes, HPLC Purified, Custom Sequence, 5' or 3' Modification |
| Catalog No. | PRIPRO-0036 |
| Description | Custom Cy5-labeled oligonucleotide probes for applications requiring far-red fluorescence detection (Ex/Em approximately 649/670 nm). Cy5, a cyanine dye with high extinction coefficient (~250,000 M^-1 cm^-1) and good quantum yield, is conjugated to either the 5'-terminus (via amino modifier C6 phosphoramidite during synthesis) or the 3'-terminus (via Cy5 CPG solid support). Each probe is HPLC-purified to >=95% full-length product and verified by MALDI-TOF mass spectrometry. Cy5-labeled oligonucleotides are widely used in microarray hybridization, fluorescence in situ hybridization (FISH), single-molecule fluorescence spectroscopy, FRET-based assays (Cy5 as acceptor), and multiplex qPCR where Cy5 provides a distinct channel well-separated from FAM, VIC/HEX, and ROX/Texas Red. The long emission wavelength of Cy5 minimizes autofluorescence background from biological samples. |
| Intended Use | Fluorescence detection probes for: microarray-based gene expression and genotyping (spotted and Affymetrix-type arrays requiring Cy5); fluorescence in situ hybridization (FISH) for chromosomal and single-gene localization; multiplex qPCR as the 4th or 5th detection channel; single-molecule FRET (smFRET) as acceptor dye paired with Cy3 donor; flow cytometry detection of labeled oligonucleotides; DNA-protein binding assays (electrophoretic mobility shift, fluorescence anisotropy); molecular beacon probes; DNA nanostructure and origami technology. |
| Principle / Technology | Cy5 (indodicarbocyanine) is a fluorescent dye with absorption maximum at 649 nm and emission maximum at 670 nm. It is conjugated to the oligonucleotide via a stable amide bond formed between Cy5-NHS ester and a 5'-amino modifier (C6 linker) on the oligonucleotide, or is directly attached to the 3'-end during solid-phase synthesis using Cy5-functionalized controlled-pore glass (CPG). HPLC purification separates the full-length Cy5-conjugated product from uncoupled oligonucleotide, free dye, and truncated synthesis byproducts, ensuring homogeneous probe population with consistent fluorescence per molecule. |
| Detection Method | Customer provides sequence (5' to 3', 15-50 bases preferred); specify 5'-Cy5, 3'-Cy5, or internal Cy5-dT modification. Synthesis: oligonucleotide assembled by standard phosphoramidite chemistry on automated synthesizer; amino modifier C6 added at 5'-end (for 5'-Cy5) or Cy5 CPG used (for 3'-Cy5); post-synthesis: Cy5-NHS ester conjugated to 5'-amino group in sodium bicarbonate buffer pH 8.5, 4 h at RT; HPLC purification on C18 column with triethylammonium acetate/acetonitrile gradient; QC: MALDI-TOF MS, UV-Vis (A260, A649), capillary electrophoresis. Lyophilized delivery with QC documentation. |
| Sample Type | Customer-specified oligonucleotide sequence (standard IUPAC code: A, C, G, T); length 15-50 bases for optimal synthesis yield and purity; sequences with secondary structure, homopolymer runs (>6 identical bases), or extreme GC content (<30% or >70%) may have reduced yield. |
| Performance Range / Specifications | Oligonucleotide length: 15-50 bases; dye position: 5'-end, 3'-end, or internal (Cy5-dT); purity: >=95% by HPLC; dye incorporation: >=90% (ratio of A649/A260); mass accuracy: within 0.1% of calculated MW by MALDI-TOF; spectral properties: Abs max 649 +/-3 nm, Em max 670 +/-3 nm. |
| Sensitivity / LOD | Cy5-labeled oligonucleotide detectable at <1 nM concentration by standard fluorescence spectrometer; single-molecule detection achievable with TIRF or confocal microscopy setups; qPCR detection: Cy5 channel sensitivity comparable to FAM/VIC in most real-time PCR instruments. |
| Specificity | Cy5 conjugation is site-specific (5'-amino, 3'-CPG, or internal amino-dT); no cross-labeling of other positions; no detectable free dye co-eluting with probe peak by HPLC; probe sequence specificity determined by customer design and bioinformatic verification. |
| Reaction Conditions / Protocol | Cy5 optimal fluorescence at pH 7.0-8.5; photostability: Cy5 is susceptible to photobleaching under intense illumination; use anti-fade mounting media for FISH and minimize light exposure during handling. |
| Components / Formulation | Lyophilized Cy5-labeled oligonucleotide (specified nmol quantity: 5 nmol, 10 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, or 1 umol); QC Documentation: HPLC chromatogram, MALDI-TOF mass spectrum, UV-Vis absorption spectrum, Certificate of Analysis. |
| Storage Conditions | Lyophilized: stable at -20 C protected from light (amber tube or foil wrap) for 24 months; reconstituted (100 uM in TE): stable at -20 C protected from light for 12 months; avoid repeated freeze-thaw cycles. |
| Shelf Life | 24 months from date of synthesis at -20 C lyophilized. |
| Package Specifications | 5 nmol, 10 nmol, 25 nmol, 50 nmol, 100 nmol, 250 nmol, 1 umol (lyophilized). |
| Product Form | Blue lyophilized pellet (Cy5 is intensely blue); reconstitution yields blue solution; Cy5 fluorescence is not visible to the naked eye (far-red). |
| Quality Control | Each probe: (1) RP-HPLC purity >=95% (single dominant peak, >=85% area); (2) MALDI-TOF MS (observed mass within 0.1% of calculated); (3) UV-Vis spectrum (A260, A649 — dye incorporation >=90%); (4) Capillary electrophoresis (>=95% full-length product); (5) Fluorescence emission spectrum (Em max 670 +/-3 nm); (6) Quantification by A260 (extinction coefficient calculated from base composition). |
| Key Features | Far-red Cy5 fluorophore (Ex/Em 649/670 nm); HPLC purified >=95%; MALDI-TOF mass confirmed; 5', 3', or internal labeling; high extinction coefficient (~250,000 M^-1 cm^-1); suitable for multiplex and single-molecule applications; custom sequence. |
| Purity | >=95% full-length Cy5-labeled product by HPLC and CE; free dye <1%; dye incorporation >=90% of oligonucleotides. |
| Concentration | Lyophilized: specified nmol; reconstitution to 100 uM in TE recommended; working concentration application-dependent: 10-500 nM for most fluorescence assays. |
| Activity / Unit Definition | Cy5 fluorescence quantum yield approximately 0.27 in aqueous buffer; extinction coefficient approximately 250,000 M^-1 cm^-1 at 649 nm; brightness (extinction coeff. x quantum yield) approximately 67,500 M^-1 cm^-1. |
| Molecular Weight | Calculated from sequence composition plus dye contribution (Cy5 adds approximately 654 g/mol for NHS ester conjugate, 632 g/mol for CPG). |
| Source / Origin | Oligonucleotide synthesized by automated phosphoramidite chemistry; Cy5-NHS ester from commercial supplier; HPLC-grade acetonitrile and triethylammonium acetate; all reagents RNase-free grade. |
| pH Range / Optimal pH | Cy5 fluorescence stable and maximal at pH 6.5-8.5; fluorescence decreases below pH 5.0; probe stable at pH 6.0-9.0. |
| Shipping Conditions | Ambient temperature; lyophilized probes stable at ambient for 4 weeks; protect from light during transit (amber tube). |
| Expiration Date / Stability | 24 months lyophilized at -20 C; 12 months in TE (100 uM) at -20 C protected from light; working dilution stable at 2-8 C for 1 month protected from light. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Cy5 detection: compatible with most microarray scanners (635 nm laser, 670 nm emission filter), fluorescence microscopes with Cy5 filter cube (Ex 590-650 nm, Em 660-740 nm), and real-time PCR instruments with Cy5 channel (Ex 620-650 nm, Em 660-700 nm). Not compatible with instruments lacking far-red detection. For qPCR, pair with FAM, VIC/HEX, ROX/Texas Red, and Quasar 705 for 5-plex detection. Cy5 signal may be reduced in the presence of high concentrations of divalent cations (>10 mM Mg2+, >5 mM Ca2+). Compatible with formamide, dextran sulfate, Denhardt's solution, and other standard hybridization buffers. |
| Recommended Buffer System | Reconstitute in nuclease-free TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) or nuclease-free water; avoid phosphate buffers at >50 mM (absorbance at 649 nm may be affected). |
| Application Notes / Precautions | Cy5 is susceptible to photobleaching; all handling steps should be performed under dim light or amber-filtered light. Upon receipt, centrifuge briefly, reconstitute to 100 uM in TE, vortex, spin down, and store at -20 C in the dark. Determine actual concentration by NanoDrop: dilute 1:10 in TE, measure A260 (correct for Cy5 contribution to A260 approximately 0.05 x A649), calculate concentration using sequence-specific extinction coefficient. Cy5 exhibits cis-trans isomerization under prolonged illumination, causing fluorescence blinking; this is a known photophysical property and should be accounted for in single-molecule experiments. For FISH, Cy5 is compatible with DAPI counterstaining (DAPI: Ex/Em 358/461 nm, no spectral overlap with Cy5). |
| Batch-to-Batch Consistency | HPLC purity >=95% for every synthesis; MALDI-TOF mass within 0.1% of calculated; dye incorporation >=90% (A649/A260 ratio). |
For research use only, not for clinical use.
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