The Best qPCR Dye - EvaGreen®
Many PCR dyes, such as SYBR® Green I, require an optimal dye concentration to avoid interference with the PCR reaction that can be quite low limiting the PCR signal, which 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 produces several products.
Cambridge Bioscience offers the high performance, stable, non-mutagenic, non-cytotoxic green fluorescent DNA binding dye, EvaGreen® from Biotium. EvaGreen® is ideal for qPCR, DNA melt curve analysis, isothermal amplification, capillary gel electrophoresis, DNA quantification in solution and selective detection of dead cells in cell viability tests. This dye is compatible with instruments equipped a 488nm argon laser or visible light excitation.
The Best qPCR Dye - EvaGreen®
The EvaGreen® dye is constructed of two monomeric DNA-binding dyes linked by a flexible spacer. In the absence of DNA, the dye assumes a non-binding looped conformation. When DNA is available, this looped conformation shifts to a random conformation that is capable of binding to DNA to emit fluorescence.
The chemical equilibrium provides a unique mechanism to continuously supply the active form of the dye from the "reserve" as more DNA is formed during a PCR process. Consequently, an EvaGreen® master mix can be formulated with relatively high dye concentration to maximise fluorescence signal without PCR inhibition, making the mix suitable for both qPCR and HRM applications. Moreover, the EvaGreen® dye in the mix is sufficiently concentrated to serve as a DNA gel stain such that PCR product can be directly analyzed by gel electrophoresis without the need for another gel stain.
Benefits of Using EvaGreen®
• Superior for qPCR & isothermal amplification
Exhibit much less PCR inhibition than other PCR dyes via a smart "release-on-demand" DNA-binding technology
• Low PCR inhibition
Low PCR inhibition of the dye permits a higher dye concentration to be used for much greater qPCR and melt curve analysis signals (Practicing HRM may require a license from Idaho Technologies, Inc)
• Nonmutagenic, noncytotoxic & environmentally safe
Nonmutagenic and noncytotoxic by standard Ames test and designed to be completely impermeable to cell membranes
• Thermostable, hydrolytically stable & photostable
Alternative DNA binding dyes have been shown to degrade following multiple freeze-thaw cycles and under PCR conditions
• Spectrally similar to other PCR dyes
Compatible with all major brand qPCR instruments and enzyme systems
• Compatible with fast PCR Protocol
Minimal interference to PCR makes it possible to significantly shorten the chain extension time
• Compatible with multiplex PCR
No dye migration from amplicon to amplicon when used at the recommended concentration
Cambridge Bioscience offers a range of products using EvaGreen®:
1. Fast EvaGreen® qPCR Master Mix
Fast Evagreen® qPCR Master Mix is a PCR reagent suitable for qPCR using a fast cycling protocol. The reagent system uses both the EvaGreen® qPCR dye and the Cheetah Taq hotstart DNA polymerase, to deliver superior results when compared to other commercial master mixes.
2. Fast-Plus EvaGreen® qPCR Master Mix
The Fast-Plus EvaGreen® qPCR master mix is a ready-to-use hot-start mix for nucleic acid quantitation and melt curve analysis of PCR amplicons. This master mix contains the safe EvaGreen® dye and the fast-activating chemically-modified hotstart enzyme Cheetah™ Taq. EvaGreen® binds to dsDNA via a novel “release-on-demand” mechanism permiting saturation dye concentration in qPCR without PCR inhibition. This mix is available in three formats containing different amounts of ROX reference dye for use in different PCR instruments.
Note: availability depends on country - see product detail page.