HOT FIREPol® EvaGreen® qPCR Mix Plus (no ROX)

Solis BioDyne's 5x ready-to-use master mix is designed for real-time quantitative PCR assays and incorporates the EvaGreen® DNA-binding dye. The mix contains all components needed to perform qPCR, including HOT FIREPol® DNA Polymerase, ultrapure dNTPs, MgCl2 and EvaGreen® dye, so the user only needs to add water, template and primers. This format is supplied without a ROX reference dye. The polymerase is activated by a 12-minute incubation at 95°C, which prevents extension of non-specifically annealed primers and primer-dimers formed at low temperatures during reaction setup.

The 5x buffer provides 12.5 mM MgCl2, equivalent to 2.5 mM in the 1x PCR solution. Recommended applications include detection and quantification of DNA and cDNA targets, gene expression profiling, microbial detection, and viral load determination. EvaGreen® is described as a DNA-binding dye with much less PCR inhibition than SYBR® Green I, extreme stability, and a non-mutagenic, non-cytotoxic profile, and is compatible with common real-time PCR cyclers using standard SYBR® Green or FAM settings.

The recommended 20 µl reaction uses 4 µl of the 5x mix, forward and reverse primers at 80–250 nM each, and variable template; suggested template ranges are 0.1 pg/µl–10 ng/µl for cDNA and 10 pg/µl–4 ng/µl for gDNA. The cycling protocol comprises an initial 95°C step for 12 minutes followed by 40 cycles of 15 s at 95°C, 20 s at 60–65°C, and 20 s at 72°C, with annealing and extension times extendable to 30 s for templates longer than 150 bp. Routine storage is -18°C to -28°C, with shipping and up to one month at room temperature stated to have no detrimental effect on quality. The product is manufactured by Solis BioDyne in Tartu, Estonia under an ISO 9001 and ISO 13485 certified Quality Management System. For research use only; not for use in diagnostic procedures.