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

A 5x ready-to-use master mix from Solis BioDyne designed for real-time quantitative PCR (qPCR) using the EvaGreen® intercalating dye. The formulation contains HOT FIREPol® DNA Polymerase, ultrapure dNTPs, MgCl2 and EvaGreen® dye, so the user only needs to add water, template and primers. The 5x buffer supplies 12.5 mM MgCl2, giving 2.5 mM MgCl2 at a 1x reaction concentration. The polymerase is activated by a 12 minute incubation at 95 °C, which limits extension of non-specifically annealed primers and primer-dimers formed at low temperatures during reaction setup, which is performed at room temperature.

Typical 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 low PCR inhibition, high stability and reported non-mutagenic, non-cytotoxic properties, and it is compatible with all common real-time PCR cyclers when the standard SYBR® Green or FAM settings are selected. The recommended 20 µl reaction uses 4 µl of the 5x mix with 80–250 nM of each primer, with suggested template inputs of 0.1 pg/µl–10 ng/µl for cDNA and 10 pg/µl–4 ng/µl for gDNA. A standard cycling protocol consists of an initial 95 °C activation for 12 min, followed by 40 cycles of 95 °C denaturation for 15 s, 60°–65 °C annealing for 20 s and 72 °C extension for 20 s; for templates longer than 150 bp the annealing and extension steps may be extended to 30 s.

The product is supplied for research use only and is not intended for use in diagnostic procedures. Routine storage is at -18 °C to -28 °C, and shipping or temporary storage for up to 1 month at room temperature is reported to have no detrimental effect on quality. It is manufactured by Solis BioDyne under an ISO 9001 and ISO 13485 certified Quality Management System.