HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX)

Manufactured by Solis BioDyne, this 5x ready-to-use master mix is designed for real-time quantitative PCR, incorporating EvaGreen® dye. The mix contains all the components required to perform qPCR, so the user needs only to add water, template, and primers. Its components include HOT FIREPol® DNA Polymerase, ultrapure dNTPs, MgCl2, EvaGreen® dye, and ROX dye, and it is compatible with both low ROX and high ROX system requirements. The MgCl2 is supplied at 12.5 mM, giving 2.5 mM in a 1x PCR solution.

HOT FIREPol® DNA Polymerase is activated by a 12-minute incubation at 95°C, which helps prevent extension of non-specifically annealed primers and primer-dimers formed at low temperatures during reaction setup. A recommended 20 µl reaction uses 4 µl of the 5x mix, with forward and reverse primers at 80–250 nM final concentration, and template cDNA in the range 0.1 pg/µl–10 ng/µl or gDNA at 10 pg/µl–4 ng/µl. EvaGreen® is described as a DNA-binding dye that is extremely stable, has much less PCR inhibition than SYBR® Green I, and has been shown to be non-mutagenic and non-cytotoxic; it is compatible with all common real-time PCR cyclers.

The recommended cycling protocol begins with 12 minutes at 95°C for initial activation, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°–65°C for 20 s, and extension at 72°C for 20 s. The product is intended for applications including detection and quantification of DNA and cDNA targets, gene expression profiling, microbial detection, and viral load determination. It is for research use only and not for use in diagnostic procedures, and is manufactured under an ISO 9001 and ISO 13485 certified quality management system. Routine storage is at -18°C to -28°C; shipping or temporary storage for up to one month at room temperature has no detrimental effect on quality.