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EvaGreen® Dye & Master Mixes for qPCR

A Next-Generation DNA Binding Dye

EvaGreen® dye is a next-generation DNA-binding dye with features ideal for use in quantitative real-time PCR (qPCR) and many other applications. Biotium scientists designed the dye by taking into consideration several essential dye properties relevant to PCR, including PCR inhibition, safety, stability and fluorescence spectra of the dye. The result of our efforts is a dye superior to SYBR® Green I and other commercial PCR or high-resolution melt curve (HRM) dyes.*

In addition, EvaGreen® dye is non-toxic, non-mutagenic, and not hazardous to aquatic life.

Figure 1. EvaGreen® dye binds to dsDNA via a "release-on-demand" mechanism.
Figure 1. EvaGreen® dye binds to dsDNA via a “release-on-demand” mechanism.
Figure 2. EvaGreen® is non-toxic, non mutagenic and non hazardous to aquatic life.

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EvaGreen® Product Line – View All EvaGreen® Products


  • Environmentally safe
    Non-mutagenic, non-cytotoxic and safe to aquatic life for safe handling and easy disposal down the drain.
  • Superior for qPCR and isothermal amplification
    Far brighter than SYBR® Green I for detecting amplification due to its novel “release-on-demand” DNA-binding mechanism.
  • Unrivaled DNA melt curve performance
    Low PCR inhibition permitting use of saturation dye concentration for maximal signal and high-resolution DNA melt analysis.*
  • Serving both as a qPCR dye and a DNA gel stain
    Electrophoretically separated PCR product visualized directly via a UV box without the need for another gel stain.
  • Compatible with multiplex PCR
    Lack of dye migration from amplicon to amplicon enabling detection of multiple PCR products by melt curves.
  • Extremely stable
    Stable during storage or under PCR conditions.
  • Useful for other applications
    Currently the only qPCR dye to be used in droplet digital PCR (ddPCR). Also can be used for isothermal amplification, microfluidic PCR systems, capillary gel electrophoresis and more.

PCR Performance:

A PCR dye emits fluorescence by forming a dye-DNA complex. The interaction with DNA inevitably leads to some PCR interference. PCR inhibition by the dye can be particularly serious at the early stage of PCR, where the dye-to-DNA ratio is high. On the other hand, a sufficient dye concentration is important for generating good signal. Thus, an optimal dye concentration must be used in order to attain reliable PCR performance.

For many current DNA-binding PCR dyes, such as SYBR® Green I, the optimal dye concentration can be quite low, which limits PCR signal and also makes the dyes unsuitable for high-resolution melt curve (HRM) analysis. Furthermore, a master mix with low SYBR® Green concentration may fail to detect multiple amplicons by melt peaks due to dye migration from small amplicons to large amplicons, giving the false result of a clean single amplicon for a PCR that may, in effect, produce several products.

Figure 2. Comparison between Fast EvaGreen® master mix (green) from Biotium and two fast SYBR® Green master mixes from two leading companies, company A (blue) and company Q (red) showing superior sensitivity of EvaGreen® dye compared to SYBR® Green. A fragment of ATPG was amplified from varying amounts of human genomic DNA (200 ng to 20 pg) using an ABI 7900 Fast thermocycler.

EvaGreen® DNA-binding dye is designed using a novel concept of DNA binding via “release-on-demand” mechanism (Figure 1). The dye is constructed of two monomeric DNA-binding dyes linked by a flexible spacer. In the absence of DNA, the dimeric dye assumes a looped conformation that is inactive in DNA binding. When DNA is available, the looped conformation shifts via an equilibrium to a random conformation that is capable of binding to DNA to emit fluorescence. This chemical equilibrium provides a unique mechanism to continuously supply the active form of the dye from the “reserve” (i.e., the dye in looped conformation), as more DNA is formed during a PCR process. Consequently, an EvaGreen® master mix can be formulated with relative high dye concentration to maximize fluorescence signal without PCR inhibition, making the mix suitable for both qPCR and HRM applications (Figure 2).*

Dye Safety:

Another major advantage of EvaGreen® dye over other PCR and HRM dyes is its safety. EvaGreen® dye is the first and only PCR dye to date designed to be environmentally safe.

Very few PCR dyes have been thoroughly studied for their safety despite the increasing use of PCR in research and diagnostics and the fact that DNA-binding dyes are inherently dangerous due to their potential to cause mutation.

Thus, handling and disposal of PCR master mixes can be a health and environmental issue. Indeed, SYBR® Green I is found to be even more environmentally toxic than ethidium bromide, one of the best known mutagens. SYBR® Green I has been suggested to interfere with DNA repair mechanisms in cells, and as a result it potentiates genotoxicity of chemicals as well as DNA damage by UV light.

Although no safety data are available on other PCR and HRM dyes (e.g., SYTO9, LC Green, BRYT Green and ResoLight), those dyes are all known to enter cells in a matter of minutes, thus posing potential genotoxicity risk. With this in mind, Biotium’s scientists designed EvaGreen® dye to be cell membrane impermeable by increasing the molecular size and charge of the dye (Figure 3). Because EvaGreen® dye is denied the chance to interact with genomic DNA in living cells, it is made much safer than the other dyes. Independent laboratory tests have confirmed that EvaGreen® is nonmutagenic, noncytotoxic and safe to aquatic life. The dye has passed environmental hazardous waste regulation in the state of California (CCR title 22) for easy disposal down the drain. See the EvaGreen Safety Report for more information.

Dye Stability:

EvaGreen® dye is very stable both during storage and under PCR conditions. EvaGreen dye has been reported to be stable at temperatures as high as 65°C for up to 6 months of use in the field for real-time PCR (Mil Med. 2014 179(6):626-32).

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. Thus, when assessing the performance of an EvaGreen-based master mix, you can eliminate the stability of the dye as a variable.

Spectral Compatibility:

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.

Other applications:

EvaGreen® dye is currently the only qPCR dye to be used in droplet digital PCR (ddPCR).

EvaGreen® dye has been applied in numerous other applications, such as Fluidigm® microfluidic PCR systems, isothermal amplification, capillary gel electrophoresis, DNA quantitation in solution and selective detection of dead cells in cell viability tests. Biotium offers EvaGreen® stand-alone dye, EvaGreen® qPCR master mixes, and other reagents and kits for qPCR.

Forget-Me-Not™ EvaGreen® qPCR Master Mix:

Biotium offers several EvaGreen® Master Mixes for qPCR and melt-curve analysis. For the best combination of value and sensitivity, we suggest our new Forget-Me-Not™ Fast EvaGreen® Master Mix. Forget-Me-Not™ is a hot-start EvaGreen® dye-based master mix for use in real time PCR applications and DNA melt curve analysis. Biotium’s master mixes feature Cheetah™ Taq, our proprietary chemically-modified hot-start DNA polymerase.

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. 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 3, left). Then, when you add template containing Forget-Me-Not™  Template Buffer to the reaction, the color turns dark blue (Figure 3, right). The 2X Forget-Me-Not™ Master Mix also can be used without the Template Buffer if you prefer. Learn more…

Forget-Me-Not qPCR Master mix
Figure 3. Forget-Me-Not™ allows you to see at a glance whether you’ve added reaction components to your tubes or wells. Left: 1X Forget-Me-Not™ Master Mix; Right: Forget-Me-Not™ Master Mix after addition of Forget-Me-Not™ Template Buffer.


EvaGreen Dye & Master Mixes Products

Product NameCatalog NumberFeatures
Forget-Me-Not qPCR Master Mix31041EvaGreen dye-based master mix with a unique two-color system for tracking which reagents you've added to your reaction.
Forget-Me-Not qPCR Master Mix with ROX31042EvaGreen dye-based master mix with a unique two-color system for tracking which reagents you've added to your reaction, with ROX reference dye.
Forget-Me-Not Universal Probe Master Mix31043High-performance master mix for fluorescent probe-based PCR applications, including quantitation and SNP genotyping.
Forget-Me-Not Universal Probe Master Mix with ROX31044High-performance master mix for fluorescent probe-based PCR applications, including quantitation and SNP genotyping. With ROX reference dye.
* Practicing HRM may require a license from Idaho Technologies, Inc.

Biotium’s EvaGreen® dye technologies are covered by EvaGreen dye and applications are covered under US patent nos. 7,803,943 and 7,776,567 and pending international patents. To inquire about licensing opportunities, please contact us at