EvaGreen Manual

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1 EvaGreen 20X
Glowing Products for ScienceTM
3159 Corporate Place
Hayward, CA 94545
www.biotium.com
Product Information
Last updated: September 27, 2010
EvaGreen Dye, 20X in water
Catalog Number: 31000
Packaging Size: 5 x 1 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 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 months 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
EvaGreen dye is a green fluorescent nucleic acid dye with features
that make the dye useful for several applications including qPCR1,2, melt curve
analysis3, 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. EvaGreen dye 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 EvaGreen dye 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 “dye redistribution” problems, which can occur with SYBR
Green I during post-PCR DNA melt curve analysis. Dye redistribution problems
may make SYBR Green I unreliable for DNA melt curve analysis (Giglio, et
al. Nucleic Acid Res. 31(22), e136(2003); Wittwer, et al. Clin. Chem. 49(6),
853(2003)). On the other hand, EvaGreen dye is optimal for both qPCR and
melt curve analysis, 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, chemicallymodified 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 SYBR Green I.
Recommended Protocol for 50 µL-sized Reactions (employing
non-hotstart Taq):
1 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 EvaGreen 4
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.
2 Perform real-time PCR reaction on a thermocycling fluorometer and record
the fluorescence signal at the annealing or extension step.
1 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 the KCl concentration
and upward adjust Tris concentration.
3 The optimal Mg2+ concentration for PCR with EvaGreenTM dye is 2.5 mM.
4 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.
5 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.
2 EvaGreen 20X
Toxicity
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 membrane-impermeable (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 DNAbinding molecules by wearing protective gears. For more information on the Ames
test result, you may download a complete report at Biotium website.
Disposal
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.
Stability Comparison of EvaGreen Dye and SYBR Green I
Comparison of Cell Membrane Permeability
between EvaGreen Dye and SYBR Green I
A) 5 min. incubation
B) 30 min. incubation
SYBR Green I, 1.2 µM EvaGreen dye, 1.2 µM
SYBR Green I, 1.2 µM EvaGreen dye, 1.2 µM
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.
Figure 1. Excitation (left) and emission (right) spectra of EvaGreenTM dye
bound to dsDNA in pH 7.3 PBS buffer.
Spectral Characteristics
Wavelength (nm)
E
xc ita
tio
n E
m is si on 1. Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76 (2007).
2. Novak, et al. An integrated fluorescence detection system for lab-on-a-chip applications. Lab Chip 7, 27(2007).
3. 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).
4. 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).
5. Wang, et al. DNA quantification using EvaGreen and a real-time PCR instrument. Anal. Biochem. 356, 303 (2006).
6. Ihrig, et al. Application of the DNA-specific dye EvaGreen for the routine quantification of DNA in microplates. Anal. Biochem. 359, 265 (2006).
7. Sang, et al. Genetic mutation analysis by CE with LIF detection using inverseflow derivatization of DNA fragments. Electrophoresis 27, 3846 (2006).
8. Sang, et al. Capillary electrophoresis of double-stranded DNA fragments using a
new fluorescence intercalating dye EvaGreen. J. Sep. Sci. 29, 1275 (2006).
9. 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).
References
Biotium products are high-quality reagents and materials intended for research purposes only.
Some products are potentially hazardous chemicals - please read the Material Safety Data
Sheet for additional information regarding handling potentially hazardous chemicals. Several
of Biotium products and product applications are covered by U.S. and international patents and
pending patents. EvaGreen® dye and applications are covered under patent US patent nos.
7,803,943 and 7,776,567 and pending international patents. Our products are not available for
resale or other commercial uses without a specific agreement from Biotium, Inc. We welcome
inquiries about licensing the use of our dyes, trademarks or technologies. Please submit
inquiries by e-mail to btinfo@biotium.com. EvaGreen is a registered trademark of Biotium, Inc.
** SYBR is a registered trademark of Invitrogen, Inc.
*** Practicing real-time PCR may require additional licensing from Roche or Applied
Biosystems.
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.

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