HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX)

A 5x ready-to-use qPCR master mix from Solis BioDyne for dye-based real-time quantitative PCR assays incorporating EvaGreen® dye. The mix contains HOT FIREPol® DNA Polymerase, ultrapure dNTPs, MgCl2, EvaGreen® dye and an internal reference based on ROX dye, so only water, template and primers need to be added by the user. The product is compatible with both low ROX and high ROX system requirements.

Suitable for standard cycling applications, with uses including detection and quantification of DNA and cDNA targets, gene expression profiling, microbial detection and viral load determination. The 5x EvaGreen® qPCR buffer provides 12.5 mM MgCl2, giving 2.5 mM MgCl2 in a 1x PCR solution. HOT FIREPol® DNA Polymerase is activated by a 12 minute incubation step at 95°C, preventing extension of non-specifically annealed primers and primer-dimers formed at low temperatures during reaction setup.

The recommended cycling protocol uses an initial activation at 95°C for 12 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60–65°C for 20 seconds and extension at 72°C for 20 seconds; for templates longer than 150 bp, annealing and extension times may be increased to 30 seconds. EvaGreen® dye has much less PCR inhibition than SYBR® Green I, is extremely stable, and has been shown to be non-mutagenic and non-cytotoxic, and is compatible with all common real-time PCR cyclers using standard SYBR® Green or FAM settings. Routine storage is at -18°C to -28°C, and shipping or temporary storage for up to 1 month at room temperature has no detrimental effects on quality. The product is manufactured under ISO 9001 and ISO 13485 certified quality management and is intended for research use only, not for use in diagnostic procedures.