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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.

Product Attributes

Reference dye

High ROX, Low ROX

  • 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
Catalog #
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

Note: This product is for users who do not require template tracking. For 2-color tracking, please see kit cat# 31046, Forget-Me-Not™ EvaGreen® qPCR Master Mix (2-Color Tracking), which also contains the 40X Template Buffer component. When the 40X Template Buffer is added to your DNA template samples, they turns dark blue, allowing you to see which tubes/master mixes you subsequently add template to.

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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