Fluorescein Passive Reference Dye is specially formulated for use on Bio-Rad real-time PCR instruments such as the MyiQ, iCycler iQ, and iQ5. This inert dye may be added to quantitative, real-time PCR reactions containing SYBR® Green I. Fluorescein allows the collection of Dynamic Well Factors from the experimental plate, which is the preferred method. This normalizes the well-to-well differences that may occur due to artifacts such as pipetting errors or instrument limitations.
Fluorescein Passive Reference Dye is composed of a 500nM solution of fluorescein in 10mM Tris-HCl (pH 8.6), 0.1mM EDTA, and 0.01% Tween®-20. Generally, the optimum final fluorescein concentration is 10nM but a titration between 5-20nM may be necessary depending on the particular instrument, the amount of SYBR® in the reaction mix, and the desired background. See specific instrument instructions for further details on well factor collection. The following shows current recommendations from USB.
Fluorescein Final Concentration:
Amount per 50 μl reaction: 1.0 μl (0.5-2.0 μl)
Final Concentration: 10nM (5-20nM)
Dilution Factor: 50X
Subtract the volume of the dye from the volume of water needed to prepare the PCR reaction. | 
: ≈ 495 nm