Probe qPCR Master Mix (2x) is a universal solution for quantitative real-time PCR and two-step real-time RT-PCR and can be used on most real-time PCR cyclers available. The master mix contains Perpetual Taq DNA Polymerase (hot start), optimized reaction buffer, dNTPs (dTTP is partially replaced with dUTP). Use of the “hot start” enzyme prevents extension of misprimed products and primer-dimers during reaction setup leading to higher specificity and sensitivity of PCR reactions. The polymerase enables convenient room temperature reaction setup. Probe qPCR Master Mix (2x) contains dUTP, which partially replaces dTTP. It allows the optional use of a uracil-N-glycosylase (UNG) to prevent carryover contamination between reactions. UNG removes uracil from any dU-containing contaminating amplicons, leaving abasic sites and making DNA molecules susceptible to hydrolysis during the initial denaturation step. The use of ROX passive reference dye is necessary for all real-time PCR cyclers from Applied Biosystems and optional for cyclers from Stratagene. ROX compensates for variations of fluorescent signal between wells due to slight differences in reaction volume and fluorescence fluctuations. ROX is not involved in PCR reaction and does not interfere with real-time PCR on any instrument.