Many real-time thermal cyclers require an internal reference dye for fluorescent signal normalization and correction of well-to-well optical variations. ROX passive reference dye allows PCR reagents that do not contain ROX to be used on all ROX-dependent real-time systems. Since excitation optics of different instrument platforms vary, the optimal reference dye concentration must be matched to the given real-time cycler. This dye has been formulated as a 50x concentrated stock solution for use on the ABI PRISM 7000, 7300, 7700, or 7900 systems. For instruments that use 580–585 nm excitation for passive reference, such as the Stratagene Mx4000, Mx3000P, or Mx3005P, or the ABI PRISM 7500 sequence detection system, the ROX passive reference dye should be treated as a 750x concentrated solution. An internal reference is not required for use with any Bio-Rad real-time detection system.