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

A 5x ready-to-use qPCR master mix from Solis BioDyne, formulated with EvaGreen® dye for dye-based real-time quantitative PCR. The mix contains all components required 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. The polymerase is activated by a 12 min incubation step at 95°C, which helps prevent extension of non-specifically annealed primers and primer-dimers formed at low temperatures during reaction setup.

The 5x EvaGreen® qPCR buffer provides 12.5 mM MgCl2, giving 2.5 mM MgCl2 in the final 1x PCR solution. EvaGreen® is described as a DNA-binding dye with much less PCR inhibition than SYBR® Green I, high stability, and a non-mutagenic and non-cytotoxic profile; it is compatible with common real-time PCR cyclers using standard SYBR® Green or FAM settings. The recommended primer concentration is 80–250 nM in a total reaction volume of 20 µl, and reactions may be assembled at room temperature.

Documented applications include detection and quantification of DNA and cDNA targets, gene expression profiling, microbial detection and viral load determination. The suggested cycling protocol uses an initial activation of 95°C for 12 min, followed by 40 cycles of 95°C for 15 s, then annealing at 60°C–65°C for 20 s and extension at 72°C for 20 s. Routine storage is at -18°C to -28°C; shipping and temporary storage for up to 1 month at room temperature has no detrimental effect on quality. It is supplied for research use only, is not for use in diagnostic procedures, and is manufactured under ISO 9001 and ISO 13485 certified quality management systems.