Cyanine3 phosphoramidite, 5′-terminal

Cyanine3 Phosphoramidite, 5′: Overview, Applications, and Usage

Cyanine3 (Cy3) is a widely used fluorophore in molecular biology, ideal for labeling oligonucleotides for detection in various assays. With an excitation maximum at 555 nm and an emission at 570 nm, Cy3 emits bright yellow fluorescence. Cyanine3 phosphoramidite, 5′ is designed for easy incorporation into oligonucleotides during synthesis, making it perfect for creating 5′-Cy3-labeled sequences.

Key Features of Cyanine3 Phosphoramidite, 5′:

• Bright Fluorescence: Cy3 has an excitation maximum of 555 nm, an emission maximum of 570 nm, and a molar extinction coefficient of 150,000 L⋅mol⁻¹⋅cm⁻¹. Its fluorescence quantum yield of 0.31 ensures strong signal output.
• Correction Factors: With CF260 at 0.04 and CF280 at 0.09, Cy3 provides accurate measurements during labeling.
• High Compatibility: Cy3 phosphoramidite works with various synthesizers, making it versatile for automated workflows. It’s great for TaqMan and Molecular Beacon probes used in real-time PCR.

Applications of Cyanine3 Phosphoramidite, 5′:

1. Oligonucleotide Labeling: Cy3 is used to label the 5′ end of oligonucleotides, enhancing detection in assays like sequencing and hybridization.
2. Synthesis of Labeled Probes: It is essential for making fluorescent probes and primers, boosting sensitivity in multiplex real-time PCR.
3. Multiplex Real-Time PCR: Cy3-labeled probes work well in the TAMRA channel, allowing for simultaneous target detection.
4. Microarray Assays: Cy3-labeled oligonucleotides are ideal for detecting specific DNA or RNA sequences in microarray and hybridization assays.

Usage Guidelines:

• Condensation: Run the reaction for 3 minutes. Use 0.02 M iodine during oxidation to protect the dye.
• Deprotection: Use 30% aqueous ammonium at room temperature. For labile groups, keep deprotection under 2 hours below 55°C. For quicker deprotection, mix 30% ammonium with 40% methylamine for 10 minutes at 65°C.
• Deoxyguanidine Groups: For dimethylformamidine-protected groups, use 30% ammonium at 65°C for 2 hours. For isobutyryl-protected groups, deprotect at room temperature for 24-36 hours.

Summary

Cyanine3 phosphoramidite, 5′ is an excellent tool for creating bright, fluorescent oligonucleotides used in various molecular biology applications. With its easy integration, strong fluorescence, and reliable performance, Cy3 enhances the sensitivity and accuracy of assays like real-time PCR, hybridization, and microarrays. Proper handling and usage ensure the best results in fluorescence-based experiments.