HOT FIREPol® EvaGreen® qPCR Supermix

A 5x concentrated, ready-to-use solution for real-time quantitative PCR, the HOT FIREPol® EvaGreen® qPCR Supermix from Solis BioDyne incorporates EvaGreen® dye and contains all the components required to perform highly sensitive qPCR — excluding template and primers. These include HOT FIREPol® DNA Polymerase, an optimised buffer, a dNTP blend containing dUTP that allows optional UNG carryover-prevention treatment (with UNG supplied separately), an internal reference based on ROX dye, a GC-enhancer and a blue visualisation dye for easy pipetting. The 5x mix provides 12.5 mM MgCl2, giving 2.5 mM in the 1x reaction. The hot-start polymerase is activated by a 12-minute incubation at 95 °C, preventing extension of non-specifically annealed primers and primer-dimers formed during reaction setup at low temperatures.

Typical applications include detection and quantification of DNA and cDNA targets, gene expression profiling, microbial detection and viral load determination. The recommended 20 µl reaction uses 100–200 nM each of forward and reverse primer, with cDNA template at 0.1 pg/µl–10 ng/µl or gDNA at 10 pg/µl–4 ng/µl. Cycling conditions are an initial 95 °C activation for 12 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60–65 °C for 20–30 s, with longer hold times used for templates above 150 bp. The supermix is compatible with all common real-time PCR cyclers using low-ROX settings; it is not compatible with high-ROX instruments such as the Applied Biosystems StepOne or StepOnePlus, for which a separate ROX formulation is recommended by the manufacturer.

Routine storage is at -18 °C to -28 °C until the end of shelf life, and shipping or temporary storage for up to one month at room temperature has no detrimental effect on product quality. The supermix is manufactured by Solis BioDyne under an ISO 9001 and ISO 13485 certified quality management system and is supplied for research use only.