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Forget-Me-Not™ EvaGreen® qPCR Master Mix (2-Color Tracking)

Forget-Me-Not™ EvaGreen® is a high performance qPCR master mix with 2-color reaction component tracking system available for a fraction of the cost of similar products.

Product Attributes

Reference dye

No ROX, With Separate ROX

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 master mix with 2-color reaction component tracking system, at a great price

  • High performance qPCR master mix at a fraction of the cost, as low as $20/mL
  • 2-color system helps you catch pipetting mistakes, saving time, samples, and $$$
  • EvaGreen® Dye – more sensitive than SYBR® Green, HRM-compatible, safer & more environmentally friendly
  • Cheetah™ Hotstart Taq for fast cycling protocols
  • Separate ROX for use with any PCR instrument

Forget-Me-Not™ EvaGreen® qPCR Master Mix is a hot-start EvaGreen® dye-based master mix for use in real time PCR applications and DNA melt curve analysis. Forget-Me-Not™ features a unique combination of a master mix containing a low concentration of blue dye, plus a DNA template buffer containing a higher concentration of blue dye. When you add the 2X Forget-Me-Not™ Master Mix to your reaction, it appears light blue (Figure 1, left). Then, when you add template containing Forget-Me-Not™ Template Buffer to the reaction, the color turns dark blue (Figure 1, right). Forget-Me-Not™ allows you to see at a glance whether you forgot to add master mix or template to any of your reactions, so you can catch pipetting mistakes and avoid wasting time, reagents, and your precious DNA samples.

Note: The Forget-Me-Not™ Master Mix can be used without the Template Buffer. If you do not require 2-color tracking, you can also use cat# 31045/31046 Forget-Me-Not™ EvaGreen® qPCR Master Mix (Low ROX or High ROX), which are premixed with ROX at concentrations optimized for instruments requiring either high or low concentrations of ROX reference dye.

Left: 1X Forget-Me-Not™ Master Mix. Right: 1X Forget-Me-Not™ Master Mix after addition of Forget-Me-Not™ Template Buffer.
Left: 1X Forget-Me-Not™ Master Mix. Right: 1X Forget-Me-Not™ Master Mix after addition of Forget-Me-Not™ Template Buffer.

Kit Components:

Forget-Me-Not™ EvaGreen® qPCR Master Mix
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 distinguish wells containing reaction mix from empty wells. You can use the master mix with or without the Forget-Me-Not Template Buffer.

Forget-Me-Not™ Template Buffer
The 40X Template Buffer contains a high concentration of inert blue dye. Adding the template buffer to your DNA template samples allows you to easily track which reactions have had template added when your are setting up your PCR reactions.

ROX Passive Reference Dye
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.
The Forget-Me-Not™ qPCR Master Mix (2-Color Tracking) is not premixed with ROX dye. Product #31041 is supplied without ROX, while product #31042 is supplied with a separate vial of ROX. Download the Product Information sheet (PI-31041, 31042) for instructions of how to prepare reactions/master mixes containing optimal ROX concentrations. We also sell Forget-Me-Not™ EvaGreen® qPCR master mixes that are premixed with ROX at low (cat# 31045) and high ROX concentrations (cat# 31046).


EvaGreen® dye
A unique DNA-binding dye with features ideal for both qPCR and High Resolution Melting® (HRM) analysis (Ref. 3). 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 (Ref. 1).

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 being 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 (Ref. 2). 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 hot-start 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.

Peace of mind, plus PCR performance
Forget-Me-Not™ qPCR Master Mix has been formulated for fast cycling PCR parameters, but can also be used with regular cycling protocols. Forget-Me-Not™ qPCR Master Mix performs as well as our Fast EvaGreen® Master Mix, and as well as or better than Qiagen’s QuantiNova® SYBR® Green PCR Master Mix in a real-time PCR assay (Figure 2), while making it easy for you to make sure your reactions are set up correctly the first time, every time.

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 mixes that are premixed with ROX at low (cat# 31045) and high ROX concentrations (cat# 31046).
**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 Taq are covered under US and international patents. QuantiNova is a registered trademark of Qiagen Group. SYBR is a registered trademark of Thermo Fisher Scientific. HRM is a registered trademark of Idaho Technologies, Inc./BioFire Defense, LLC; the use of HRM may require a license.



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