NZY DNA Ligase Master Mix (2x) is a premixed ready to use solution that embodies an ultrapure recombinant enzyme specialized in catalysing the formation of phosphodiester bonds between abutting 5′-phosphate and 3′-hydroxyl termini in duplex DNA or RNA structures. In addition, it was meticulously formulated with buffer, additives and cofactors to improve both blunt and cohesive ends overhang substrates’ ligation and transformation into Escherichia coli competent cells, thus expediting experimental workflows and reaction set-up. This master mix is formulated with an optimal enzyme/buffer ratio to facilitate efficient, reliable, and consistent ligations in a timeframe of seconds to 60 minutes at ambient laboratory conditions (20-25 °C). Outstandingly, this master mix can streamline and enhance the efficiency of challenging molecular biology projects, with the possibility to directly transform ligation products into competent cells without prior dilution.