ROX Passive Reference Dye
ROX™ Passive Reference Dye is specially formulated for use on Applied Biosystems (ABI) and Stratagene real-time PCR instruments. This inert dye, whose fluorescence does not change during the reaction, may be added to quantitative, real-time PCR reactions to normalize the well-to-well differences that may occur due to artifacts such as pipetting errors or instrument limitations.
ROX Passive Reference Dye is composed of a 25μM solution of 5-carboxy-X-rhodamine in 10mM Tris-HCl (pH 8.6), 0.1mM EDTA, and 0.01% Tween®-20. Although many instruments require use of the dye at final concentrations of 500nM, newer instruments with optimized filter sets require use at 50nM. See specific instrument instructions for further details on passive dye usage. The following shows current recommendations from USB.
ROX™ Final Concentration for Different Instruments:
ABI 7000, 7300, 7700, 7900HT and 7900HT Fast:
Amount per 50 μl reaction: 1.0 μl (0.6-1.0 μl)
Final ROX Concentration: 500nM (300-500nM)
Dilution Factor: 50X
ABI 7500 and ABI 7500 Fast; Stratagene Mx3000™, Mx3005P™, and Mx4000™:
Amount per 50 μl reaction: 0.1 μl (0.06-0.1 μl)*
Final ROX Concentration: 50nM (30-50nM)
Dilution Factor: 500X
Subtract the volume of the dye from the volume of water needed to prepare the PCR reaction.
*Note: ROX may be diluted 1:10 in water to aid in accurate pipetting.
Spectral Characteristics:
Excitation Maximum λ: ≈ 575 nm
Emission Maximum λ: ≈ 600 nm
Storage:
Shipped on dry ice. Store at -20°C. Protect from light.