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EvaGreen® Dye and Master Mixes

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).

For most qPCR applications, we recommend using EvaGreen® Dye, 20X  in water (31000), or one of our 2X EvaGreen® qPCR Master Mixes. EvaGreen® Dye, 2000X  in DMSO (31019) is offered for specialized applications requiring a concentrated dye solution.

If necessary, EvaGreen® Dye, 2000X  in DMSO can be diluted in water or master mix for qPCR for a final concentration of 1X in the PCR reaction.

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 compared to SYBR® Green-based master mixes. You can read your signal in the green fluorescence channel (FAM, SYBR® Green, or EvaGreen®) of your instrument.

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.

Forget-Me-Not™ EvaGreen® qPCR Master Mix is a hot-start EvaGreen® dye-based master mix. It uses Biotium’s proprietary chemically modified hot-start DNA Polymerase, Cheetah™ Taq. This master mix also contains a light blue tracking dye, and comes with a dark blue template dye, for two-color tracking to reduce pipetting errors. The user only needs to supply primers and template.

Forget-Me-Not™ Universal Probe Master Mix is formulated for fluorescent probe-based PCR applications. It uses Biotium’s proprietary chemically modified hot-start DNA Polymerase, Cheetah™ Taq. The user needs to supply some kind of fluorescent probe (such as TaqMan®, dual-labeled BHQ®, or Molecular Beacon) in addition to primers and template.

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.

The ROX concentration is 35 nM which is suitable for low ROX instruments. If you are using a high ROX instrument, you can add ROX, 25 mM in TE Buffer (Catalog # 29052) which is available separately (Cat # 29052).

EvaGreen® Dye is supplied at 20X (25 uM) in water (cat. no. 31000) or 2000X (2.5 mM) in DMSO (cat. no. 31019). Other formulations are available for product licensees, inquire at

Also see our ready-to-use EvaGreen® Master Mixes for qPCR.

Our original EvaGreen® Dye is a saturating dsDNA binding dye that is superior for quantitative real-time PCR (qPCR), high-resolution melt (HRM), digital droplet PCR (ddPCR) and other genomics applications over SYBR® Green I and other commercial PCR dyes. It offers several essential features critical for PCR and related applications including high thermal, chemical and photostability, high sensitivity due to high signal to noise related to its novel ‘release-on-demand’ mechanism, non-inhibitory to PCR, and lack of dye migration.  In addition, EvaGreen® Dye is non-toxic, non-mutagenic, and not hazardous to aquatic life.

EvaGreen® Plus Dye is an advanced version of the original EvaGreen® Dye, retaining the same essential benefits while providing a higher signal-to-noise ratio. This greater sensitivity offers further advantages for digital PCR and isothermal applications.

EvaGreen® Dye is used in all of Biotium’s dye-based qPCR master mixes, which combine superior brightness and sensitivity, with the ability to do melt curve analysis in the same reaction. The new EvaGreen® Plus Dye is available as a stand-alone 20X solution in water.

Our qPCR Master Mixes are all offered with optional ROX reference dye, which is used by some qPCR instruments to normalize the PCR fluorescence. We also include instructions for using no ROX, low ROX, or high ROX, depending on the instrument you are using. Based on instrument manufacturers’ recommendations, we recommend the following:


BioRad: iCycler™, MyiQ™, MiQ™2, iQ™5, CFX-96 Touch™, CFX-384 Touch™, Chromo4™, MiniOpticon™

Qiagen: Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene® 6000

Eppendorf: Mastercycler® Realplex

Illumina: Eco™ RealTime PCR System

Cepheid: SmartCycler®

Roche: LightCycler® 480, LightCycler® 2.0


ABI: 7500, 7500 Fast, ViiA 7™, QuantStudio™

Stratagene: MX4000P, MX3000P, MX3005P

High ROX

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne®, StepOne Plus®

0.5X to 1X EvaGreen® is commonly used for LAMP applications when fluorescent signal is read by instrumentation. For detection by eye, an increased concentration of EvaGreen® can be used. For example,  Lee, S. H. et al. tested up to 100X EvaGreen® and found that 10X EvaGreen® was optimal for their assay.

Lee, S. H. et al. Front. Microbiol., 09 January 2017 |

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