IRDye 800-NHS

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.

IRDye 800 NHS Alternate
Fluorophore Name	Excitation Max, nm +/-10	Emission Max, nm +/-10	Extinction Coefficient*
cy7.5 NHS	788	808	223,000
IRDye 800 NHS	795	819	240,000
* 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.

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

IRDye800 is a near-IR fluorescent dye used for labeling oligonucleotides. IRDye800 has an absorbance maximum of 778 nm and an emission maximum of 794 nm. The combination of narrow absorbance/emission bands and low-background autofluorescence in the IR region results in higher S/N ratios and thus enhanced detection sensitivity compared with fluorophores with absorbance/emission maxima in the visible region (1). IRDye800 is used as a reporter moiety in real-time PCR applications. For such probes, IRDye800 is most commonly paired with the dark quencher QC-1, as the two have excellent spectral overlap (2).

IRDye800 can be used to label DNA oligos for use as hybridization probes in a variety of in vivo and in vitro research or diagnostic applications, as well as for structure-function studies of DNA, RNA, and protein-oligonucleotide complexes. Oligos labeled with IRDye800 at the 5'-end can be used as PCR and Sanger DNA sequencing primers to generate fluorescently-labeled PCR, sequencing or genetic analysis (AFLP, microsatellite) products (3-5).

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
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
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. Middendorf, L.R., Bruce, J.C., Eckles, R.D., Grone, D.L., Roemer, S.C., Sloniker, G.D., Steffens, D.L., Sutter, S.L., Brumbaugh, J.A., et al. Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus. Electrophoresis (1992), 13: 487-494.
2. Peng X., Chen, H., Draney, D.R., Volcheck, W., Schutz-Geschwender, A., Olive, D.M. A nonfluorescent, broad-range quencher dye for Forster resonance energy transfer assays. Anal. Biochem. (2009), 388: 220-228.
3. Yomano, L.P., Scopes, R.K., Ingram, L.O. Cloning, sequencing, and expression of the Zymomonas mobilis phosphoglycerate mutase gene (pgm) in Escherichia coli. J. Bacteriol. (1993), 175: 3926-3933.
4. Oetting, W.S., Lee, H.K., Flanders, D.J., Wiesner, T.A., King, R.A. Linkage Analysis with Multiplexed Short Tandem Repeat Polymorphisms Using Infrared Fluorescence and M13 Tailed Primers. Genomics (1995), 30: 450-458.
5. Myburg, A.A., Remington, D.L, O’Malley, D.M., Sederoff, R.R., Whetton, R.W. High-Throughput AFLP Analysis Using Infrared Dye-Labeled Primers and an Automated DNA Sequencer. Biotechniques (2001), 30: 348-357.
- IRDye 800-NHS