EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means no change in optical settings is required for using an EvaGreen®-based master mix. However, there are several differences between the dyes.
Lower background fluorescence: EvaGreen® dye has less background than SYBR® Green I due to its novel “release-on-demand” DNA-binding mechanism.
Brighter and non-inhibitory: EvaGreen® is less inhibitory in the PCR reaction than SYBR® Green, permitting the use of saturating dye concentration for maximal signal and better high-resolution DNA melt analysis.
Safer and more environmentally friendly: EvaGreen® dye is the first and only PCR dye to date designed to be environmentally safe.
Dye stability: EvaGreen® dye is very stable both during storage and under PCR conditions. SYBR® Green I, on the other hand, is known to degrade following multiple freeze-thaw cycles and under PCR conditions. Moreover, decomposed SYBR® Green I is reported to be even more inhibitory to PCR.
Digital PCR: EvaGreen® dye is currently the only qPCR dye to be used in droplet digital PCR (ddPCR).
Biotium offers three different EvaGreen® dye-based qPCR master mixes:
Forget-Me-Not™ qPCR Master Mix
Fast EvaGreen® qPCR Master Mix
Fast Plus EvaGreen® qPCR Master Mix
All three master mixes contain our high-quality EvaGreen® DNA-binding dye and our fast-activating chemically-modified hotstart enzyme, Cheetah™ Taq.
Forget-Me-Not™ qPCR Master Mix is our newest master mix formulation. For most purposes this is the master mix that we would recommend, as it has many benefits: best sensitivity and signal, blue tracking dyes in the master mix and template to reduce pipetting errors, and low cost. Fast EvaGreen® qPCR Master Mix is a sensitive, general use master mix that does not contain blue tracking dyes. Fast Plus EvaGreen® qPCR Master Mix has a slightly different formulation than the regular Fast EvaGreen® qPCR Master Mix, and was designed for more challenging samples.
Both of these qPCR master mixes contain Cheetah™ Taq (our chemically modified hotstart DNA polymerase), buffer and dNTPs.
The Fast EvaGreen® Master Mixes also contain the DNA-binding dye EvaGreen®, and are used for fluorescent dye-based qPCR. The only thing the user needs to supply are primers and template.
The Fast Probe Master Mix is formulated for conducting real-time PCR using an oligo-based probe, such as a TaqMan™ probe, an MGB probe or an AllGlo™ probe. The user needs to supply primers, probe and template.
High performance master mix with 2-color reaction component tracking system, at a great price
High performance qPCR master mix at a fraction of the cost of similar products, as low as $20/mL
2-color system helps you catch pipetting mistakes, saving time, reagents, DNA samples, and $$$
EvaGreen® Dye – better signal than SYBR® Green, safer and more environmentally friendly
Cheetah™ Hotstart Taq – fully activated after two minutes at 95°C for fast cycling protocols
Separate ROX for optimizing with any PCR instrument
Forget-Me-Not™ EvaGreen® qPCR Master Mix is a hot-start EvaGreen® dye-based master mix for use in real time PCR applications and DNA melt curve analysis. Forget-Me-Not™ features a unique combination of a master mix containing a low concentration of blue dye, plus a DNA template buffer containing a higher concentration of blue dye. When you add the 2X Forget-Me-Not™ Master Mix to your reaction, it appears light blue (Figure 1, left). Then, when you add template containing Forget-Me-Not™ Template Buffer to the reaction, the color turns dark blue (Figure 1, right). Forget-Me-Not™ allows you to see at a glance whether you forgot to add master mix or template to any of your reactions, so you can catch pipetting mistakes and avoid wasting time, reagents, and your precious DNA samples. The 2X Forget-Me-Not™ Master Mix also can be used without the Template Buffer if you prefer.
Forget-Me-Not™ EvaGreen® qPCR Master Mix 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 distinguish wells containing reaction mix from empty wells. You can use the master mix with or without the Forget-Me-Not™ Template Buffer.
Forget-Me-Not™ Template Buffer The 40X Template Buffer contains a high concentration of inert blue dye. Adding the template buffer to your DNA template samples allows you to easily track which reactions have had template added when your are setting up your PCR reactions.
ROX Passive Reference Dye ROX 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.
The Forget-Me-Not™ qPCR Master Mix (2-Color Tracking) is not premixed with ROX dye. Product #31041 is supplied without ROX, while product #31042 is supplied with a separate vial of ROX. Download the Product Information sheet (PI-31041, 31042) for instructions of how to prepare reactions/master mixes containing optimal ROX concentrations. We also sell Forget-Me-Not™ EvaGreen® qPCR master mixes that are premixed with ROX at low (cat# 31045) and high ROX concentrations (cat# 31046).
EvaGreen® dye A unique DNA-binding dye with features ideal for both qPCR and High Resolution Melting® (HRM) analysis*. EvaGreen® dye binds to dsDNA via a novel “release-on-demand” mechanism, which permits the use of a relatively high dye concentration in qPCR without PCR inhibition (Ref. 1).
A unique feature of EvaGreen® dye is its safety. DNA-binding dyes are inherently dangerous due to their potential to cause mutation. With this in mind, Biotium’s scientists designed EvaGreen® dye such that it cannot cross cell membranes, thus preventing the dye from being in contact with genomic DNA in live cells. All other commercial PCR dyes enter into cells in a matter of minutes. SYBR® Green I, for example, has been shown to be environmentally more toxic than ethidium bromide, a well-known mutagen (Ref. 2). Independent labs have confirmed that EvaGreen dye is nonmutagenic, noncytotoxic and safe to aquatic life for direct disposal in the drain. For details, download the EvaGreen® Dye Safety Report.
An added benefit of an EvaGreen®-based master mix is that you can analyze your PCR product by gel electrophoresis without the need to add another DNA-binding dye to either your loading buffer or gel. The EvaGreen® dye in the master mix can act as a DNA pre-stain, permitting direct visualization of DNA bands following electrophoresis.
Cheetah™ HotStart Taq 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.
Peace of mind, plus PCR performance Forget-Me-Not™ qPCR Master Mix has been formulated for fast cycling PCR parameters, but can also be used with regular cycling protocols. Forget-Me-Not™ qPCR Master Mix performs as well as our Fast EvaGreen® Master Mix, and as well as or better than Qiagen’s QuantiNova® SYBR® Green PCR Master Mix in a real-time PCR assay (Figure 2), while making it easy for you to make sure your reactions are set up correctly the first time, every time.
*Instruments that do not require ROX reference dye are generally compatible with qPCR master mixes containing ROX (check with the manufacturer before use). We also sell Forget-Me-Not™ EvaGreen® qPCR master mixes that are premixed with ROX at low (cat# 31045) and high ROX concentrations (cat# 31046).
**Bio-Rad’s iCycler™ users do not need to add fluorescein to the PCR reaction as EvaGreen® dye has a slight background fluorescence that provides adequate and stable baseline level fluorescence. For these instruments we recommend using Forget-Me-Not™ EvaGreen® qPCR Master Mix without ROX (cat# 31041).
EvaGreen® Dye and Cheetah™ HotStart Taq DNA Polymerase are covered under US and international patents. QuantiNova® is a registered trademark of Qiagen Group. SYBR® Green is a registered trademark of Molecular Probes, Inc./Life Technologies. *HRM® is a registered trademark of Idaho Technologies, Inc./BioFire Defense, LLC and its use may require a license.
1. Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76-91 (2007).
2. Ohta, et al. Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mutation Res. 492, 91-97 (2001).
2X Forget-Me-Not™ Universal Probe qPCR Master Mix is a high-performance product for fluorescent probe-based PCR applications, including quantitation and SNP genotyping. This kit is suitable for all fluorescent probe-based technologies, including hydrolysis probes (such as TaqMan® and dual-labeled BHQ® probes) and displacement probes (like Molecular Beacons).
Cheetah™ Taq is a chemically modified hot-start Taq DNA polymerase useful for preventing or reducing nonspecific DNA amplification in PCR. Modified using a novel modifying reagent, Cheetah™ Taq represents a major improvement upon AmpliTaq® Gold by having a faster activation time and better shelf-life.
EvaEZ™ Fluorometric Polymerase Activity Assay Kit provides an easy and accurate way to determine the activity of a nucleic acid polymerase without using radioisotopes. It contains EvaGreen® dye together with a primed template, dNTPs, and MgCl2 in a Tris buffer system. Available without magnesium by special order.
GelRed® is an ultra sensitive, extremely stable and environmentally safe fluorescent nucleic acid dye designed to replace the highly toxic ethidium bromide (EB) for staining dsDNA, ssDNA or RNA in agarose gels or polyacrylamide gels. GelRed® is far more sensitive than EB without requiring a destaining step.
GelGreen® is a sensitive, stable and environmentally safe green fluorescent nucleic acid dye designed to stain either dsDNA, ssDNA or RNA in agarose gels. GelGreen® is far more sensitive than SYBR Safe.