Supreme NZYProof 2× Green Master Mix is a premixed ready-touse solution containing Supreme NZYProof DNA polymerase (MB283), an engineered highly accurate, fast and sensitive variant of NZYProof DNA polymerase displaying a hot-start like PCR capacity. This feature is achieved by a novel hot-start technology, which inhibits both polymerase and 3’→5’ exonuclease activities and thus avoids extension of non-specifically annealed primers or primer-dimers, as well as the degradation of primers and template DNA during PCR reaction setup. Thus, Supreme NZYProof DNA polymerase generates higher specificity, sensitivity and yield during the accurate amplification of DNA. The master mix contains dNTPs, reaction buffer and additives at optimal concentrations for the efficient amplification of DNA templates by PCR. Mg²⁺ final concentration is 1.5 mM, allowing the implementation of a variety of PCR protocols. In addition, reactions assembled with Supreme NZYProof 2× Green Master Mix may be directly loaded onto agarose gels. Supreme NZYProof 2× Green Master Mix contains two dyes (blue and yellow) that allow monitoring the progress of electrophoresis. Supreme NZYProof 2× Green Master Mix is not suitable when direct fluorescent or absorbance readings are required without prior purification of the amplified DNA from PCR. We recommend the purification of the PCR products using NZYGelpure (MB011) before employing nucleic acids in downstream protocol. Supreme NZYProof DNA polymerase possesses 3´→5´ exonuclease proofreading activity. Resulting PCR products have blunt-ends and are suitable for cloning with NZYtech´s NZY-blunt PCR cloning kit (MB121).