Supplemental Microbial qPCR Mastermix (ROX) is highly suited for real-time PCR applications using 5′ hydrolysis probe-based detection that uses ROX as a reference dye. This supplemental mastermix is for use in automated pipetting systems that require more mastermix in addition to the mastermix supplied with the Microbial DNA qPCR Arrays and Assay Kits. Supplemental Microbial qPCR Mastermix (ROX) contains real-time PCR buffer, a high-performance HotStart DNA Taq polymerase, nucleotides, and ROX dye for optimization of the instrument optics. The chemically-modified and tightly controlled HotStart enzyme uniquely provides accurate 5′ hydrolysis probe results by preventing the amplification of primer-dimers and other nonspecific products.