AZDye-700 NHS

Click here for a list of fluorophores.

This modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C3, C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

YIELD
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.

~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis.
~30 nmol final yield for 2 umol scale synthesis.
~75 nmol final yield for 5 umol scale synthesis.
~150 nmol final yield for 10 umol scale synthesis.
~225 nmol final yield for 15 umol scale synthesis.

AZdye-700 NHS has the same chemical structure and spectral properties as of Alexa Fluor® 700 (Alexa Fluor® is the trademark of ThermoFisher).

Human vision is insensitive to light beyond ~650 nm; it is not possible to view near-IR fluorescent dyes.

AZDyes are a set of fluorescent dyes that span the visible electromagnetic spectrum, as well as some of the near-IR. The absorbance range is 346-749 nm, and the emission range is 442-775 nm. Generally speaking, AZDyes are brighter, chemically more stable, and less pH-sensitive than other fluorescent dyes commonly used to label oligonucleotides (1). Because they currently only are in the form of NHS esters, oligos first must be synthesized with an Amino Linker modification (either at the ends or internally). The appropriate AZDye is then manually attached to the oligo through the amino group in a separate reaction post-synthesis. The list of currently available dyes includes AZDye 350, -405, -430, -488, -500, -514, -532, -546, -555, -568, -594, -610, -633, -647, -660, -680, -700, -750, with the number indicating the appropriate absorbance wavelength for the particular dye. AZDyes are suitable for a variety of in vitro and in vivo applications. However, for in vivo experiments, users should note that dyes 350/405, being "blue" dyes, require higher-energy excitations than the other. Users of these particular dyes should confirm that the higher-energy required for excitation does not damage the relevant cells or tissues being used in the in vivo experiments.

-

Near Infrared Fluorophore Spectral Data & Quencher Selection Guide
Fluorophore Name	Excitation Max, nm +/-10	Emission Max, nm +/-10	Extinction Coefficient*	Color**	Quencher
Cy5	650	665	250,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
IRDye 650 NHS	650	665	230,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
AZ647 NHS	655	680	191,800	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
AZ680 NHS	678	701	185,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
Cy5.5	684	710	198,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
IRDye 700 NHS	684	710	288,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
AZdye700 NHS	696	719	192,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
Atto 700 NHS	699	715	120,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
Cy7 NHS	740	773	199,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
IRDye 750 NHS	756	776	260,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
cy7.5 NHS	788	808	223,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
IRDye 800 NHS	795	819	240,000	Human vision is insensitive to light beyond ~650 nm.	BBQ-650 λ (max) = 650nm Range = 550-750 nm
* Extinction coefficient at λ (max) in cm-1M-1. ** Typical emission color seen through the eyepiece of a conventional fluorescence microscope with appropriate filters. Near-IR region. Human vision is insensitive to light beyond ~650 nm; it is not possible to view near-IR fluorescent dyes. Click here for a list of fluorophores.
Click here for list of quenchers.

References
1. Didenko, V.V. DNA Probes Using Fluorescence Resonance Energy Transfer (FRET): Designs and Applications. Biotechniques (2001), 31: 1106-1121.
2. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
3. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761.
4. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.
- AZDye-700 NHS