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Cy5 phosphoramidite
Two cyanine derivatives, Cy3 and Cy5 phosphoramidites, are the most popular fluorescent labeling reagents for preparing fluorescence-labeled oligonucleotides. Cyanine dyes are normally added once at the 5'-terminus and the MMT group should be removed on the synthesizer. The absorbance of the MMT cation (yellow) is noticeably different from the DMT cation (orange), thus the trityl absorbance can be used to monitor oligo coupling. Cyanine dye phosphoramidites have also been used successfully adjacent to the 3'-terminus. Deprotection of oligos containing cyanine dyes may be carried out with ammonium hydroxide at room temperature, regardless of the base protecting groups on the monomers used. If ammonium hydroxide is used at elevated temperature, Cy3 and Cy3.5 are more stable than Cy 5 and Cy 5.5. However, it is always prudent to use monomers with base labile protecting groups to limit the exposure time to 2 hours or less at 65°C during deprotection.
Calculators
Common stock solution preparation
Table 1. Volume of MeCN needed to reconstitute specific mass of Cy5 phosphoramidite to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 102.076 µL | 510.381 µL | 1.021 mL | 5.104 mL | 10.208 mL |
| 5 mM | 20.415 µL | 102.076 µL | 204.152 µL | 1.021 mL | 2.042 mL |
| 10 mM | 10.208 µL | 51.038 µL | 102.076 µL | 510.381 µL | 1.021 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (260 nm) | Correction Factor (280 nm) | Correction Factor (482 nm) | Correction Factor (565 nm) |
| Cy5 tertrazine [Cy5 tertrazine] | 651 | 670 | 2500001 | 0.271, 0.42 | 0.02 | 0.03 | 0.009 | 0.09 |
| Cy5 tyramide | 651 | 670 | 2500001 | 0.271, 0.42 | 0.02 | 0.03 | 0.009 | 0.09 |
| Cy5 tetrazine | 651 | 670 | 2500001 | 0.271, 0.42 | 0.02 | 0.03 | 0.009 | 0.09 |
| Cy5 aldehyde | 651 | 670 | 2500001 | 0.271, 0.42 | 0.02 | 0.03 | 0.009 | 0.09 |
| DBCO-Cy5 | 651 | 670 | 2500001 | 0.271, 0.42 | 0.02 | 0.03 | 0.009 | 0.09 |
| Cy3 phosphoramidite | 555 | 569 | 1500001 | 0.151 | 0.07 | 0.073 | - | - |
| VIC phosphoramidite | 526 | 543 | - | - | - | - | - | - |
References
View all 1 references: Citation Explorer
Fluorescent DNA: the development of 7-deazapurine nucleoside triphosphates applicable for sequencing at the single molecule level
Authors: Seela F, Feiling E, Gross J, Hillenkamp F, Ramzaeva N, Rosemeyer H, Zulauf M.
Journal: J Biotechnol (2001): 269
Authors: Seela F, Feiling E, Gross J, Hillenkamp F, Ramzaeva N, Rosemeyer H, Zulauf M.
Journal: J Biotechnol (2001): 269
Page updated on November 21, 2024
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