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| Product Name | Amino-Modifier C6-dT Oligonucleotides, Desalted and HPLC Options, Custom Sequence |
| Catalog No. | PRIPRO-0037 |
| Description | Custom oligonucleotides containing an amino-modifier C6-dT (5'-dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) at specified internal positions, synthesized with a primary amino group on a 6-carbon linker arm attached to the C5 position of thymidine. The amino group serves as a reactive handle for post-synthetic conjugation to: N-hydroxysuccinimide (NHS) ester-activated fluorophores, biotin-NHS, haptens (digoxigenin, dinitrophenol), photocrosslinkers, quenchers (DABCYL, BHQ), spin labels, and other functional moieties. The C6 linker provides adequate spacing between the oligonucleotide backbone and the conjugated moiety to minimize steric hindrance during hybridization. This building block is essential for preparing custom fluorescent probes, affinity probes, and functionalized oligonucleotides for diagnostic assay development, biosensor construction, and chemical biology applications. |
| Intended Use | Amine-functionalized oligonucleotide intermediate for customer-conjugated: fluorescent probes (FAM, Cy3, Cy5, TAMRA, ROX, Alexa Fluor dyes via NHS ester conjugation); biotinylated capture probes for streptavidin-based affinity capture and detection; enzyme-conjugated probes (horseradish peroxidase, alkaline phosphatase); hapten-labeled probes (digoxigenin, DNP) for immunohistochemistry and in situ hybridization; photocrosslinking probes (aryl azide, benzophenone conjugation) for DNA-protein interaction mapping; surface immobilization on amine-reactive slides, beads, and biosensor chips (NHS-activated, epoxide, aldehyde surfaces). |
| Principle / Technology | The C6-dT phosphoramidite building block contains a trifluoroacetyl (TFA)-protected primary amine attached via a 6-carbon linker to the C5 position of thymidine. It is incorporated into the oligonucleotide at the desired position during standard automated DNA synthesis with extended coupling time (3-6 minutes vs standard 30-60 seconds). After synthesis, the TFA protecting group is removed during standard ammonium hydroxide deprotection (55 C, 8-16 h), revealing the free primary amine (pKa approximately 9-10). At pH 8-9, the amino group is partially deprotonated and reactive toward NHS esters, isothiocyanates, and sulfonyl chlorides for subsequent conjugation. |
| Detection Method | Customer specifies: oligonucleotide sequence, position(s) of amino-modifier C6-dT incorporation (indicate as 'X' or '[AmC6dT]'), synthesis scale (50 nmol-1 umol), and purification (desalted or HPLC). Synthesis: automated phosphoramidite synthesis with extended coupling (3-6 min) at C6-dT positions; standard TFA deprotection during NH4OH cleavage/deprotection; desalted by gel filtration or HPLC purified (C18 or anion exchange); QC: UV quantitation, MALDI-TOF MS, and optionally amine reactivity test (TNBS or ninhydrin assay). |
| Sample Type | Customer-specified sequence containing one or more positions designated as '[AmC6dT]'; length: 10-60 bases (optimal 15-40); avoid amino-dT at 3'-terminal position (may reduce conjugation efficiency and purification recovery). |
| Performance Range / Specifications | Synthesis scale: 50 nmol, 200 nmol, 1 umol; incorporation efficiency per C6-dT: >95%; overall purity (desalted): >85% full-length; overall purity (HPLC): >95% full-length; amine content: >90% of theoretical by amine-specific assay. |
| Sensitivity / LOD | Amino-oligonucleotide detection: TNBS (2,4,6-trinitrobenzenesulfonic acid) assay produces colored product detectable at 420 nm; conjugation efficiency monitored by HPLC shift or gel shift assay; as little as 1 nmol amino-oligonucleotide sufficient for standard NHS ester conjugation. |
| Specificity | The amino group is site-specifically located on the C5 position of the modified thymidine base; C6 linker provides adequate spacing from the DNA backbone; no cross-reactivity with other nucleobases under standard conjugation conditions (pH 8.5, aqueous/organic co-solvent). No detectable reaction with unmodified nucleotides under NHS ester conjugation conditions. |
| Reaction Conditions / Protocol | Oligonucleotide synthesis: standard cycles with extended coupling at C6-dT positions; deprotection: concentrated NH4OH, 55 C, 8-16 h; amine conjugation (user-performed): dissolve amino-oligo in 0.1 M sodium bicarbonate pH 8.5, add NHS ester dye/biotin in DMSO or DMF (10-20 molar excess), incubate 2-4 h at RT or overnight at 4 C; purify conjugate by ethanol precipitation, size exclusion, or HPLC. |
| Components / Formulation | Amino-modified oligonucleotide (lyophilized, specified scale and purification); QC Documentation: HPLC chromatogram (if HPLC-purified), MALDI-TOF mass spectrum, UV quantitation, Certificate of Analysis, conjugation protocol guide. |
| Storage Conditions | Lyophilized: -20 C for 24 months; reconstituted in nuclease-free water or TE (100 uM) at -20 C for 12 months; avoid exposure to amine-reactive contaminants (aldehydes, CO2 from air causing carbamate formation); store under inert gas (argon or nitrogen) if possible. |
| Shelf Life | 24 months from date of synthesis at -20 C. |
| Package Specifications | 50 nmol, 200 nmol, 1 umol (desalted); 50 nmol, 200 nmol (HPLC purified). |
| Product Form | White to off-white lyophilized pellet (sodium salt); reconstitution yields clear colorless solution. |
| Quality Control | (1) OD260 quantification; (2) MALDI-TOF mass spectrometry (mass within 0.1% of calculated); (3) HPLC or CE purity: desalted >85%, HPLC >95%; (4) Amine reactivity test (ninhydrin or fluorescamine assay positive for primary amine); (5) Specific activity: >90% of theoretical amine content by TNBS assay. |
| Key Features | C6 amino linker on dT for post-synthetic conjugation; amino group for NHS ester, isothiocyanate, and sulfonyl chloride chemistry; desalted or HPLC purification; 50 nmol to 1 umol scales; includes conjugation protocol guide; C6 spacer minimizes steric effects on hybridization. |
| Purity | Desalted: >85% full-length product; HPLC: >95% full-length product; amine content >90% of theoretical; free amine protecting group reagent <0.1%. |
| Concentration | Lyophilized: specified nmol; reconstitute to 100 uM in nuclease-free water or TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA); determine actual concentration by A260 measurement. |
| Activity / Unit Definition | Amine reactivity confirmed by fluorescamine assay (fluorescence at Ex/Em 390/475 nm) and by conjugation test with FAM-NHS ester (>85% conjugation efficiency under standard conditions). |
| Molecular Weight | Calculated from sequence composition with C6-dT modification (C6-dT adds approximately 296 g/mol relative to unmodified dT, after TFA deprotection). |
| Source / Origin | Oligonucleotide synthesized by automated phosphoramidite chemistry; Amino-Modifier C6-dT phosphoramidite from commercial supplier (Glen Research 10-1039 or equivalent); all synthesis reagents: anhydrous acetonitrile, activator (5-ethylthio-1H-tetrazole), oxidation (iodine), capping reagents. |
| pH Range / Optimal pH | Free amine pKa approximately 9-10; protonated (inactive) below pH 7; deprotonated and reactive above pH 8; conjugation optimal at pH 8.3-8.5 (sodium bicarbonate buffer); oligonucleotide stable at pH 6.0-9.0. |
| Shipping Conditions | Ambient temperature (lyophilized); protect from moisture. |
| Expiration Date / Stability | 24 months at -20 C lyophilized; reconstituted in TE pH 8.0: 12 months at -20 C under argon; amine reactivity decreases over time in solution due to oxidation and carbamate formation — for critical conjugations, use within 3 months of reconstitution. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Compatible with standard NHS ester conjugation chemistry: dye-NHS (FAM, Cy3, Cy5, TAMRA, ROX, Alexa Fluor 488/532/546/594/647), biotin-NHS, biotin-LC-NHS, photocrosslinker-NHS, hapten-NHS. Compatible with isothiocyanate conjugation (FITC, TRITC). Compatible with sulfonyl chloride chemistry (dansyl chloride). Conjugation buffer: 0.1 M sodium bicarbonate pH 8.3-8.5, or 0.1 M sodium borate pH 8.5. Avoid amine-containing buffers (Tris, glycine, ammonium) during conjugation. Amine-free organic co-solvents (DMF, DMSO up to 30% v/v) compatible for dissolving hydrophobic NHS esters. |
| Recommended Buffer System | Amino-oligo reconstitution: nuclease-free water or TE (10 mM Tris-HCl pH 7.0-8.0, 0.1-1 mM EDTA); Conjugation buffer: 0.1 M NaHCO3 pH 8.5 or 0.1 M sodium borate pH 8.5; purification: TE or water after conjugation. |
| Application Notes / Precautions | The free amine is susceptible to oxidation and reaction with ambient CO2 (forming carbamates). For long-term storage, aliquot the reconstituted amino-oligonucleotide under argon or nitrogen and store at -80 C. For conjugation: dissolve dye-NHS ester in anhydrous DMSO or DMF immediately before use (NHS esters hydrolyze rapidly in water — half-life ~30 min at pH 8.5, 25 C). Use 20-50 fold molar excess of NHS ester over oligonucleotide for efficient conjugation. After conjugation, remove excess dye by ethanol precipitation (3 volumes ethanol, 0.3 M sodium acetate pH 5.2, -20 C overnight), size exclusion chromatography (NAP-5, NAP-10, or G-25 columns), or HPLC purification. Monitor conjugation efficiency by analytical HPLC or 20% denaturing PAGE. |
| Batch-to-Batch Consistency | MALDI-TOF mass within 0.1% of calculated for every synthesis; amine content >90% of theoretical; conjugation efficiency >85% with FAM-NHS standard test. |
For research use only, not for clinical use.
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