EvaGreen® Dye, 20X in Water

A green fluorescent nucleic acid dye with features that are ideal for applications such as qPCR, HRM®, LAMP, and digital PCR.

Product Description

EvaGreen® Dye is green fluorescent nucleic acid dye that is essentially non-fluorescent by itself, but becomes highly fluorescent upon binding to dsDNA. It is ideal for a wide variety of applications including qPCR and DNA melt curve analysis, HRM®, LAMP, digital PCR, real-time monitoring of thermophilic helicase-dependent amplification (tHDA), capillary gel electrophoresis, and more. The dye is provided at 20X (25 uM) in water.

Brighter and less inhibitory than SYBR® Green
Environmentally safe, non-mutagenic, and non-cytotoxic
Unsurpassed thermal stability, chemical stability, and photostability
Excellent compatibility with multiplex PCR
Directly visualize amplification products in a gel
Compatible with common qPCR instruments and enzyme systems

The Unique Properties of EvaGreen® Dye

EvaGreen® Dye was designed with a novel “release-on-demand” mechanism. In the absence of DNA, the dye assumes a conformation that is inactive. When DNA is available, the dye shifts to an active conformation that binds DNA to emit fluorescence. This property of the dye provides a unique mechanism to continuously supply the active form of the dye from an inactive reserve, as more DNA is produced during the PCR process. As a result, EvaGreen® Dye can be used at a much higher dye concentration than SYBR® Green I (Ref. 1), resulting in a high resolution signal for digital PCR, real-time qPCR, HRM®, and multiplex qPCR by DNA melt curve analysis. This technology also makes it possible to significantly shorten the chain extension time, enabling the use of fast PCR protocols. Learn more about EvaGreen® Dye Technology.

EvaGreen® Dye Applications

When bound to dsDNA, EvaGreen® Dye has excitation and emission spectra very close to those of fluorescein (FAM) or SYBR® Green I, making it readily compatible with instruments equipped with the 488 nm argon laser or any visible light excitation with wavelength in the region. In addition to qPCR and HRM, EvaGreen® Dye is currently the only qPCR dye to be used in droplet digital PCR (ddPCR). EvaGreen® can also be used for isothermal amplification, microfluidic PCR systems, capillary gel electrophoresis, and more. EvaGreen® amplification products can be directly detected on a gel without the need for another DNA gel stain. Download a list of Selected EvaGreen® References for more information.

A Safer, More Stable PCR Dye

EvaGreen® Dye is environmentally friendly, non-mutagenic, and non-cytotoxic because it is impermeable to cell membranes, unlike SYBR® Green I, which enters cell rapidly and is known to be a powerful mutation-enhancer (Ref. 2). Download the EvaGreen® Dye Safety Report for more information. EvaGreen® Dye is more convenient to handle because it has no detectable dye decomposition in PCR buffer at 95-100°C for 48 hours; is highly stable under either alkaline or acidic condition; and withstands repeated freeze-thaw cycles.

Our Forget-Me-Not™ EvaGreen® qPCR Master Mix with ROX as well as our Forget-Me-Not™ EvaGreen® qPCR Master Mix with 2-Color Tracking utilizes EvaGreen® Dye in a convenient ready-to-use qPCR master mix with excellent sensitivity and competitive prices. We also offer Thiazole Green, 1000X in DMSO, which is identical to SYBR® Green I.

EvaGreen and applications are covered under granted and pending US and international patents. SYBR is a registered trademark of Thermo Fisher Scientific. HRM is a registered trademark of Idaho Technologies, Inc./BioFire Defense, LLC and its use may require a license.

References

BMC Biotechnol (2007) Nov 9;7:76 doi: 10.1186/1472-6750-7-76

EvaGreen® Dye in droplet digital PCR:
1. Lab Chip (2010) Oct 21;10(20):2666-72 doi: 10.1039/c004521g
2. Anal Chem (2010) 82, 6, 2310-2316 doi: 10.1021/ac902510u
3. J Am Chem Soc (2011) 133, 44, 17705-17712 doi: 10.1021/ja2060116
4. Anal Chem (2013) 85, 23, 11619-11627 doi:10.1021/ac403061n
5. Anal Chem (2013) 85, 22, 11129-11136 doi: 10.1021/ac4030413
6. Nucleic Acids Res (2013) Oct;41(18):e175 doi:10.1093/nar/gkt684
7. Analyst (2014) Jun 7;139(11):2695-701 doi: 10.1039/c3an02334f
8. Anal Chem (2014) 86, 5, 2618-2624 doi: 10.1021/ac403843j

Find a list of EvaGreen® references under Supporting Documents.

Product Attributes

Excitation/Emission	500/530 nm (with DNA)
Concentration	20X (25 uM)
Storage Conditions	Store at -10 to -35 °C

Reference Publications

Mote, R.D., Laxmikant, V.S., Singh, S.B. et al.

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

J Biosci 46, 109 (2021).

DOI: https://doi.org/10.1007/s12038-021-00231-w

Article Snippet: “Complementary DNA (cDNA) was diluted (1:10) times and used as a template for Real-time PCR. EvaGreen dye (Biotium Cat no. 31000) was used to prepare mastermix.”

Gavrilov, Momčilo et al.

Engineered helicase replaces thermocycler in DNA amplification while retaining desired PCR characteristics

Nature communications vol. 13,1 6312.

DOI: doi:10.1038/s41467-022-34076-0

Article Snippet: “This section contains information about SHARP reaction components and their concentrations. Other stock components are dNTP’s (10 mM each), ATP (100 mM), Evagreen dye (20X from Biotium # 31000), DTT (Dithiothreitol, 100 mM in water).”

Ruihua Ding, Liying Liu, Jiali Zhang, Pengxiao Lv, et al.

Accurate quantification of DNA using on-site PCR (osPCR) by characterizing DNA amplification at single-molecule resolution

Nucleic Acids Research, Volume 51, Issue 11, 23 June 2023, Page e65.

DOI: https://doi.org/10.1093/nar/gkad388

Article Snippet: “In this work, only FAM channel is used and the exposure time is set at default 1s. 1x KAPA2G buffer supplemented with 0.5 mM Mg²⁺ and 0.2 mM dNTP. 0.8x Evagreen (Cat. 31000, Biotium, California, USA) is used as DNA dye.”

Wang K, Jiang Y, Guo Y, Geng M, Wu W.

An Optimized Thermal Feedback Methodology for Accurate Temperature Control and High Amplification Efficiency during Fluorescent qPCR

Bioengineering. 2022; 9(6):237.

DOI: https://doi.org/10.3390/bioengineering9060237

Article Snippet: “The PCR reagent of the SARS-CoV-2 was composed of 1× Premix Ex Taq HS (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China), 1× EvaGreen Dye (31000, Biotium, CA, USA). Each test requires 20 µL of reagent and 15 µL mineral seal above that.”

Chua Yi Jing, Sim Steven Poh Chuen, Shridharan Medha, Seow Yiqi.

Simultaneous and rapid colorimetric detection of distinct miRNAs using Split-LAMP

Frontiers in Bioengineering and Biotechnology, VOLUME 11, 2023 ISSN=2296-4185

DOI: https://doi.org/10.3389/fbioe.2023.1271297

Article Snippet: “The fluorescence dye used for detection is EvaGreen (catalog number #31000, Biotium). The TA values are derived using the relative threshold algorithm on QuantStudio software.”

Swennenhuis, J.F., Reumers, J., Thys, K. et al.

Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS

Genome Med 5, 106 (2013).

DOI: https://doi.org/10.1186/gm510

Article Snippet: ” The proteinase K was inactivated by incubating for 10 minutes at 96°C. After this step, 5 μl of an amplification mix containing Evagreen, a double-stranded DNA dye (Biotium, Hayward, CA, USA; cat. 31000), was added to each well.”

Documents

EvaGreen® Dye, 20X in Water

EvaGreen® Dye, 2000X in DMSO

AccuBlue® Broad Range dsDNA Quantitation Kit with DNA Standards

MyGel Mini Electrophoresis System