ROX is a rhodamine dye that has played a significant role in dideoxy Sanger sequencing alongside FAM, JOE and TAMRA. The dye itself is also commonly used for qPCR applications as a passive reference dye. ROX is not available as a phosphoramidite, and as such, the use of this NHS ester is the standard method of incorporating ROX into oligonucleotides. This NHS ester is offered as the 6-isomer.
Coupling: For labeling 0.2 µmoles of an amino-modified oligonucleotide, dissolve 3.2 mg of the ROX NHS Ester in 100 µL DMSO (or DMF). Dissolve the amino-modified oligonucleotide in 500 µL 0.1 M carbonate/bicarbonate buffer (pH 9). Add 20 µL of the NHS Ester solution and vortex. Let react for 60 min at room temperature and desalt.