MasterMix is a ready-to-use reagent in which all the components are already mixed in an optimized way. Additional stabilizers are added to enable long-term storage.
The concentration of components is perfectly adapted to most of the assays.
A HotStart Taq polymerase is inactive at low temperatures (room temperature). Heating at 95 °C activates the enzyme, and the amplification can begin once the primers are annealed. The enzyme is not active until the entire DNA is denatured.
dNTPs / dUTPs
Some kits contain a blend of dNTPs and dUTPs, other ones contain only dNTPs.
The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and double-stranded DNA containing dUTPs.
Some thermocyclers require MasterMix containing ROX dye for normalization. This is the case for the ABI and Eppendorf machines, and optional on the Stratagene machines.
For some thermocyclers, the normalization method for SYBR® Green assay uses Fluorescein to create a “virtual background”.
MgCl2 is necessary for the Reverse Transcriptase and the Taq activity. MgCl2 concentration in MasterMixes is optimized according to the amount of Taq and also the buffer composition.
Inert colored dye
Some buffers also include an inert colored dye, to enable visualization of the buffer when loading in the wells. This colored dye has no effect on the sensitivity of the assay and is a convenient working tool.