EvaGreen® Dye and high-performance dye-based qPCR master mixes containing EvaGreen® qPCR dye, Cheetah™ HotStart Taq Polymerase, and Forget-Me-Not™ tracking dye. Available with 2-colour tracking or pre-mixed with low or high ROX.

• Ready-to-use, just add primers and DNA template
• Superior EvaGreen® Dye is more sensitive than SYBR® Green I and is compatible with HRM
• Prevent wasted samples and costly mistakes with Forget-Me-Not™ 2-colour tracking
• Formulated with Cheetah™ HotStart Taq for fast cycling protocols
• Directly visualise amplification product fluorescence in a gel
• No ROX or Separate ROX for use with any PCR instrument
• Available premixed with ROX and optimised for instruments requiring either high or low concentrations of ROX reference dye

2X Forget-Me-Not™ EvaGreen® qPCR Master Mix is a ready-to-use high performance dye-based master mix for qPCR and DNA melt curve analysis. The 2X Master Mix contains EvaGreen® Dye, Cheetah™ HotStart Taq DNA Polymerase, dNTPs, and a low concentration of an inert blue dye, which allows the user to easily distinguish wells containing reaction mix from empty wells. For 2-colour tracking, mix your template with Forget-Me-Not™ Template Buffer to see at a glance which wells have master mix or template. Forget-Me-Not™ EvaGreen® qPCR Master Mix is also available premixed with ROX in two formulations: a low ROX master mix (~50 nM final ROX concentration in assay) and a high ROX master mix (~500 nM final assay concentration). ROX is required as a passive reference dye in some qPCR systems to compensate for well-to-well optical variations.

The Unique Properties of EvaGreen® Dye:
EvaGreen® Dye was designed with a novel 'release-on-demand' mechanism. In the absence of DNA, the dye assumes a conformation that is inactive. When DNA is available, the dye shifts to an active conformation that binds DNA to emit fluorescence. This property of the dye provides a unique mechanism to continuously supply the active form of the dye from an inactive reserve, as more DNA is produced during the PCR process. As a result, EvaGreen® Dye can be used at a much higher dye concentration than SYBR® Green I, resulting in a high resolution signal for digital PCR, real-time qPCR, HRM®, and multiplex qPCR by DNA melt curve analysis. This technology also makes it possible to significantly shorten the chain extension time, enabling the use of fast PCR protocols.

EvaGreen® Dye Applications:
When bound to dsDNA, EvaGreen® Dye has excitation and emission spectra very close to those of fluorescein (FAM) or SYBR® Green I, making it readily compatible with instruments equipped with the 488 nm argon laser or any visible light excitation with wavelength in the region. In addition to qPCR and HRM, EvaGreen® Dye is currently the only qPCR dye to be used in droplet digital PCR (ddPCR). EvaGreen® can also be used for isothermal amplification, microfluidic PCR systems, capillary gel electrophoresis, and more. EvaGreen® amplification products can be directly detected on a gel without the need for another DNA gel stain.

A Safer, More Stable PCR Dye:
EvaGreen® Dye is environmentally friendly, non-mutagenic, and non-cytotoxic because it is cell membrane-impermeant, unlike SYBR® Green I, which enters cell rapidly and is known to be a powerful mutation-enhancer. EvaGreen® Dye is more convenient to handle because it has no detectable dye decomposition in PCR buffer at 95-100°C for 48 hours; is highly stable under either alkaline or acidic condition; and withstands repeated freeze-thaw cycles.
Highly concentrated EvaGreen® Dye, 2000X (2,5 mM) in DMSO is offered for customers with specialised applications requiring a concentrated dye solution. For most qPCR applications, we recommend our ready-to-use Forget-Me-Not™ EvaGreen® qPCR Master Mixes or EvaGreen® Dye, 20X in water. EvaGreen® Dye is a patented green fluorescent nucleic acid dye that is essentially non-fluorescent by itself, but becomes highly fluorescent upon binding to dsDNA.

Cheetah™ HotStart Taq DNA Polymerase is Biotium’s proprietary chemically-modified hot-start DNA Polymerase. Cheetah™ Taq is completely inactive at room temperature, and is fully activated after 2 minutes at 95 °C, making it particularly suitable for fast cycling PCR protocols.
ROX Reference Dye is required as a passive reference dye in some qPCR systems to compensate for well-to-well optical variations. Refer to the table below for the passive dye requirements of real-time PCR instruments. Product 31041-1, 31041-20ML, and 31041-T are supplied without ROX, while product 31042-1, 31042-20ML and 31042-T are supplied with a separate vial of ROX. For master mixes with pre-mixed ROX, use Forget-Me-Not™ EvaGreen® qPCR Master Mix (Low ROX or High ROX).