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Forget-Me-Not™ EvaGreen® qPCR Master Mix (Low ROX or High ROX)

Forget-Me-Not™ EvaGreen® qPCR Master Mix is a hotstart EvaGreen® dye-based master mix for use in real time PCR applications and DNA melt curve analysis.

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  • Fraction of the cost of similar products
  • Light blue master mix color to prevent pipetting errors
  • EvaGreen® Dye – better signal than SYBR® Green
  • Cheetah™ Hotstart Taq – fully activated after two minutes
Reference dye

High ROX, Low ROX

Reference dye
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1 mL (100 reactions) Trial Size
5 x 1 mL (500 reactions)
2 x 10 mL (2000 reactions)
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Product Description

High performance EvaGreen® master mix at a great price

  • High performance qPCR master mix at a fraction of the cost of similar products, as low as $20/mL
  • Light blue master mix color helps to prevent pipetting mistakes, saving time and reagents
  • EvaGreen® Dye – better signal than SYBR® Green, safer and more environmentally friendly
  • Cheetah™ HotStart Taq – fully activated after two minutes at 95°C for fast cycling protocols
  • Premixed with ROX at concentrations optimized for instruments requiring either high or low concentrations of ROX reference dye
Figure 1: Forget-Me-Not™ EvaGreen® qPCR Master Mix contains a low concentration of an inert blue dye, which allows the user to visually distinguish wells containing reaction mix from empty wells, and can thereby reduce pipetting errors, saving time and reagents.

The 2X Master Mix contains EvaGreen® dye, Cheetah™ HotStart Taq DNA Polymerase, dNTPs, and a low concentration of an inert blue dye, which allows the user to easily distinguish wells containing reaction mix from empty wells.

EvaGreen® dye
A unique DNA-binding dye with features ideal for both qPCR and High Resolution Melting® (HRM) analysis***. EvaGreen® dye binds to dsDNA via a novel “release-on-demand” mechanism, which permits the use of a relatively high dye concentration in qPCR without PCR inhibition (Mao, et al. 2007. BMC Biotech. 7, 76-91).

A unique feature of EvaGreen® dye is its safety. DNA-binding dyes are inherently dangerous due to their potential to cause mutation. With this in mind, Biotium’s scientists designed EvaGreen® dye such that it cannot cross cell membranes, thus preventing the dye from coming in contact with genomic DNA in live cells. All other commercial PCR dyes enter into cells in a matter of minutes. SYBR® Green I, for example, has been shown to be environmentally more toxic than ethidium bromide, a well-known mutagen (Ohta, et al. 2001. Mut. Res. 492, 91-97). Independent labs have confirmed that EvaGreen® dye is nonmutagenic, noncytotoxic and safe to aquatic life for direct disposal in the drain. For details, download the EvaGreen® Dye Safety Report.

An added benefit of an EvaGreen®-based master mix is that you can analyze your PCR product by gel electrophoresis without the need to add another DNA-binding dye to either your loading buffer or gel. The EvaGreen® dye in the master mix can act as a DNA pre-stain, permitting direct visualization of DNA bands following electrophoresis.

FMN, Fast EG, QuanitNova qPCR
Figure 2. Real-time PCR data comparing Forget-Me-Not™ (blue lines) with Biotium’s Fast EvaGreen® (green lines) and Qiagens QuantiNova® SYBR® Green (gray lines) master mixes. A. Amplification curves on linear scale. EvaGreen® dye-based master mixes yield higher signal compared to the SYBR® Green-based mix. B. Amplification curves on logarithmic scale. Forget-Me-Not™ performs as well or better than the other master mixes. NTCs: non-template controls (dashed lines).

Cheetah™ HotStart Taq
Cheetah™ HotStart Taq DNA Polymerase is Biotium’s proprietary chemically-modified hotstart DNA Polymerase. Cheetah™ Taq is completely inactive at room temperature, and is fully activated after 2 minutes at 95°C, making it particularly suitable for fast cycling PCR protocols.

Forget-Me-Not™ EvaGreen® qPCR Master Mix with ROX is available in two formulations: a low ROX master mix (~50 nM final ROX concentration in assay) and a high ROX master mix (~500 nM final assay concentration). ROX is required as a passive reference dye in some qPCR systems to compensate for well-to-well optical variations. Refer to the table below for the passive dye requirements of real-time PCR instruments.

Reference Dye PCR Instrument
(~50 nM)
Applied Biosystems®: 7500, 7500 Fast, ViiA™7, Quant-Studio™ instruments
Stratagene (Agilent): MX4000P, MX3000P, MX3005P
High ROX
(~500 nM)
Applied Biosystems®: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne™, StepOnePlus™
No ROX required*BioRad: iCycler™, MyiQ™, MiQ™ 2, iQ™ 5, CFX-96 Touch™, CFX-384 Touch™ and Connect™, Chromo4™, MiniOpticon™
Qiagen: Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene®6000
Eppendorf: Mastercycler® Realplex
Illumina: Eco™ RealTime PCR System
Cepheid: SmartCyler®
Roche: LightCycler® 480, LightCycler® 2.0
Fluorescein**BioRad: iCycler™, MyiQ™, MiQ™, iQ™
*Instruments that do not require ROX reference dye are generally compatible with qPCR master mixes containing ROX (check with the manufacturer before use). We also sell Forget-Me-Not™ EvaGreen® qPCR Master Mix kits without ROX (cat# 31041), and with a separate tube of ROX (cat# 31042) that can be used to make your own low or high ROX master mixes.
**Bio-Rad’s iCycler™ users do not need to add fluorescein to the PCR reaction as EvaGreen® dye has a slight background fluorescence that provides adequate and stable baseline level fluorescence. For these instruments we recommend using Forget-Me-Not™ EvaGreen® qPCR Master Mix without ROX (cat# 31041).

EvaGreen® Dye and Cheetah™ HotStart Taq DNA Polymerase are covered under US and international patents. QuantiNova® is a registered trademark of Qiagen Group. SYBR® Green is a registered trademark of Molecular Probes, Inc./Life Technologies.
***HRM® is a registered trademark of Idaho Technologies, Inc./BioFire Defense, LLC and its use may require a license.


1. Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76-91 (2007).
2. Ohta, et al. Ethidium bromide and SYBR Green I enhance the genotoxicity of UV-irradiation and chemical mutagens in E. coli. Mutation Res. 492, 91-97 (2001).


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Support & Faq

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).

Category: EvaGreen® dye and Master Mixes

← FAQs

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.

Category: EvaGreen® dye and Master Mixes

← FAQs

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.

Category: EvaGreen® dye and Master Mixes

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