EvaGreenTMDye, 20X in water

Catalog Number:31000

Packaging Size: 5x1 mL

Molecular Information: Proprietary*

Color and Form: Light orange solution

Spectral Property: λabs/λem = 500/530 nm (DNA bound); λabs = 471 nm (without DNA)


Storage and Handling

EvaGreen dye is generally very stable. We recommend EvaGreen 20X solution be stored at 4°C or below to prevent mold formation. The expected shelf-life under the recommended condition should be at least 12 month from the date of receipt. When taking the dye solution out of the freezer, vortex the solution for a few seconds in case of dye adsorption on the container wall during storage.

Product Description

EvaGreendye is a green fluorescent nucleic acid dye with features that make the dye useful for several applications including qPCR1,2, highresolution DNA melt curve analysis (HRM)3, real-time monitoring of thermophilic helicase-dependent amplification (tHDA)4, routine solution DNA quantification5,6 and capillary gel electrophoresis7,8. The DNA-bound dye has excitation and emission spectra very close to those of fluorescein (FAM) or SYBR Green I (Figure 1), making the dye readily compatible with instruments equipped with the 488 nm argon laser or any visible light excitation with wavelength in the region. EvaGreen dye is extremely stable both thermally and hydrolytically (Figure 2), providing convenience during routine handling. The dye is essentially nonfluorescent by itself, but becomes highly fluorescent upon binding to dsDNA. EvaGreendye is nonmutagenic and noncytotoxic by being completely impermeable to cell membranes (Figure 3), unlike SYBR Green I, which enters cell rapidly and is known to be a powerful mutation-enhancer9.

The unique properties of EvaGreendye have made it particularly useful in quantitative real-time PCR (qPCR) application. Compared with the widely used SYBR Green I, EvaGreen dye is generally less inhibitory toward PCR and less likely to cause nonspecific amplification. As a result, EvaGreen dye can be used at a much higher dye concentration than SYBR Green I, resulting in more robust PCR signal. More significantly, the higher EvaGreen concentration permitted for qPCR eliminates so-called “dye redistribution” problem, which can occur with SYBR Green I during post PCR DNA melt curve analysis. Dye redistribution problem may make SYBR Green I unreliable for regular DNA melt curve analysis (Giglio, et al. Nucleic Acid Res. 31(22), e136(2003)) and also unsuitable for HRM (Wittwer, et al. Clin. Chem. 49(6), 853(2003)). On the other hand, EvaGreen dye is suitable for both qPCR and HRM, yielding robust and reproducible results.

EvaGreen 20X solution is specifically formulated for qPCR use. PCR reaction can be monitored using your existing optical setting for SYBR Green I or FAM on any commercial real-time PCR cycler. The qPCR protocol provided below is for PCR using regular non-hot-start Taq. Use of a hot-start Taq may require some adjustment of PCR buffer composition in terms of ionic strength and pH to best take the advantage of EvaGreen dye. For example, chemically-modified Taq, such as AmpliTaq Gold, may prefer a downward-adjustment of KCl concentration (to as low as 0.0 mM) and an upward-adjustment of Tris concentration (to as high as 50 mM). In addition, a water soluble solvent such as DMSO or glycerol has traditionally been added to stabilize a master mix. These components plus the pH may need to be optimized depending on the nature of your enzyme. Nevertheless, if you use a regular non-hot-start Taq and follow the protocol provided below, you should expect to see superior performance from EvaGreen dye over that from SYBR Green I. Because the optical settings vary slightly from instrument to instrument and the wavelengths of EvaGreen dye are slightly longer than those of SYBR Green I, Ct value may differ slightly by +1 or –1 when compared with SYBR Green I side-by-side. However, regardless of which cycler you use, the fluorescence signals with EvaGreen dye for both qPCR and melt curve analysis should be significantly stronger than those with SYBRGreen I.

Recommended Protocol for 50 μL-sized Reactions (employing non-hotstart Taq):

  • Set up PCR reaction as follows1: 5 μL of 10x polymerase buffer without magnesium 2 2.5μL of 50mM MgCl2 3 5 μL each of 2 mM dNTP 2.5 μL of 20X EvaGreen4 1-5 units of Taq DNA polymerase5 0.1-1 μM each of primers (final concentrations) Add Di-H2O to make a final volume of 50 μL.
  • Perform real-time PCR reaction on a thermocycling fluorometer and record the fluorescence signal at the annealing or extension step.
    For iCycler users, you do not need to add FAM to your PCR mix since EvaGreenTM has a slight background fluorescence that provides an adequate and stable baseline level fluorescence for well calibration.
    When using ABI Sequence Detection Systems, make sure to select NONE for the passive reference under the tab WELL INSPECTOR.
    BSA may be required if the reaction is run on a Roche LightCycler. A final BSA concentration of 0.5mg/mL may be sufficient. With SYBR Green, addition of a protein such as BSA results in a fluorescence increase, which provides a background signal that triggers the start of a LightCycler. Since EvaGreenTM dye is less sensitive to proteins, you may need to adjust the instrument setting (for background fluorescence) so that the instrument will start.

    2 For chemically-modified Taq, it may be necessary to downward adjust KCl concentration and upward adjust Tris concentration.

    The optimal Mg2+ concentration for PCR with EvaGreenTM dye should be 2.5 mM.

    Before pipetting, warm up the 20X solution to room temperature and thoroughly mix the solution by vortexing. EvaGreenTM is highly stable. However, dye adsorption onto

    container wall may occur during storage at low temperature over a long period of time. Should it occur, vortexing the vial for a few seconds should alleviate the problem.

    For best results, a hot-start enzyme should be used. However, buffer condition may need to be adjusted accordingly to best take the advantage of the dye.


Ames test performed by an independent lab, Litron Laboratories (Rochester, NY), showed that EvaGreen dye is nonmutagenic as well as noncytotoxic. EvaGreen dye appears to be completely cell membraneimpermeable (Figure 3), which may be a key factor responsible for the observed low toxicity. On the other hand, SYBR Green I is known to be a powerful mutation enhancer, possibly by inhibiting the natural DNA repairing mechanism in cells (Ohta, et el. Mutat. Res. 492, 91(2001)). The toxicity of SYBR Green I may be associated with its ability to enter cells rapidly (Figure 3).

Since these toxicity tests were not performed on human, we still advise that researchers exercise precautions when handling the dye or any other DNA-binding molecules by wearing protective gears. For more information on the Ames test result, you may download a complete report at Biotium website.


EvaGreen solution may be disposed of using one of the following methods: 1) Add 25~50 mL bleach (regular household bleach) to each gallon (~4L) of the waste solution containing the dye and let the mixture react for at least 8 hours before pouring the solution to a sink; 2) Pour each 10 liters of EvaGreen waste solution through ~1g of activated charcoal. The filtrate may directly go to the drain while the charcoal may be treated as regular solid waste.


  • Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76 (2007).
  • Novak, et al. An integrated fluorescence detection system for lab-on-a-chip applications. Lab Chip 7, 27(2007).
  • White, et al. Methylation-sensitive high-resolution melt-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman Syndromes. Clin. Chem. 53(11), 1 (2007).
  • Goldmeyer, et al. Development of a novel one-tube isothermal reverse transcription thermophilic helicase-dependent amplification platform for rapid RNA detection. J. Mol. Diag. 9(5), 639 (2007).
  • Wang, et al. DNA quantification using EvaGreen and a real-time PCR instrument. Anal. Biochem. 356, 303 (2006).
  • Ihrig, et al. Application of the DNA-specific dye EvaGreen for the routine quantification of DNA in microplates. Anal. Biochem. 359, 265 (2006).
  • Sang, et al. Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments. Electrophoresis 27, 3846 (2006).
  • Sang, et al. Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen. J. Sep. Sci. 29, 1275 (2006).
  • Ohta, et el. Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mutat. Res. 492, 91 (2001).

Spectral Characteristics

Figure 1. Excitation (left) and emission (right) spectra of EvaGreenTM dye bound to dsDNA in pH 7.3 PBS buffer.

Stability Comparison of EvaGreenDye and SYBRGreen I

Figure 2. A solution of EvaGreenTM dye or SYBR Green I each at 1.2 μM in pH 9 Tris buffer was incubated at 99 oC. The absorption spectrum of each solution was followed over a period of 3 hours. ROX was added as a stable reference.

Comparison of Cell Membrane Permeability between EvaGreenDye and SYBR Green I

A) 5 min. incubation
B) 30 min. incubation
Figure 3. HeLa cells were incubated with SYBR Green I (1.2 μM) or EvaGreen dye (1.2 μM) at 37 oC. Photographs were taken following incubation for 5 min (panel A) and 30 min (panel B). SYBR Green I entered cells rapidly while EvaGreen appeared membrane-impermeable.