Supreme NZYLong 2× Green Master Mix is a premixed ready-to-use solution containing Supreme NZYLong DNA polymerase (MB331), an engineered version of NZYLong DNA polymerase designed to amplify longer PCR products, generally of 25 kb and beyond, that displays a higher specificity provided by its 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 nonspecifically annealed primers or primer-dimers, as well as degradation of primers and template DNA during PCR reaction setup. The functional activity of the enzyme is restored during a short 5-minute incubation step at 94 °C. The increased processivity of Supreme NZYLong DNA polymerase combined with the hot-start-like PCR capacity results in higher specificity, sensitivity and yield during the amplification of long nucleic acids. The master mix contains dNTPs, reaction buffer and additives at optimal concentrations for the efficient amplification of a wide range of DNA templates. MgCl₂ final concentration is 2.5 mM, allowing the implementation of a variety of PCR protocols. In addition, reactions assembled with Supreme NZYLong 2× Green Master Mix may be directly loaded onto agarose gels. There are two dyes (blue and yellow) in the mix that allow monitoring the progress of the electrophoresis. Supreme NZYLong 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 using the master mix version without dyes Supreme NZYLong 2× Colourless Master Mix (MB334) or purifying the PCR product using NZYGelpure (MB011) before performing any other protocol. Supreme NZYLong DNA polymerase generates a mixture of A-overhang-ended (predominantly) and blunt-ended PCR products, being suitable for cloning with NZYtech’s TA PCR cloning kits (MB053 or MB137).

Extension time: 60 sec/kb at 68 °C

3’→5′ activity: no

Product overhang: mixed