NZYSupreme qPCR Probe Master Mix (2x), ROX plus is an optimized and highly efficient reaction mixture developed for real-time PCR, specifically for probe-detection technology (including molecular beacons). This master mix was engineered with a dual hot-start enzyme control mechanism to provide the highest detection sensitivity. In addition, the latest developments in PCR enhancers have been incorporated in the NZYSupreme qPCR Probe Master Mix, including buffer chemistry and incorporation of highly robust engineered enzymes. The inclusion of ROX reference dye in the master mix formulation does not interfere with the fluorescence emission of the reporter dyes commonly used in real-time PCR and provides advantages in the experiment. The presence of ROX reference dye in the master mix enables to increase confidence in data analysis, since it allows to normalize non-PCR-related fluctuations in fluorescence. Despite most real-time PCR instruments that are able to read ROX dye allow users to run experiments and analyse data without ROX, the inclusion of this internal passive reference dye prevents data misinterpretation and allows to detect and diagnose errors. NZYSupreme qPCR Probe Master Mix (2x), ROX plus is provided as a simple-to-use, stabilized 2x reaction mixture that includes all components for quantitative PCR, except sample DNA, primers, probe and water.