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

A high-performance dye-based qPCR master mix containing EvaGreen®, Cheetah™ HotStart Taq Polymerase, and the Forget-Me-Not™ 2-color tracking system.

Kit Components:

  • Forget-Me-Not™ EvaGreen® qPCR Master Mix
  • Forget-Me-Not™ Template Buffer
  • ROX Passive Reference Dye (31042 cat no.’s only)
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

2X Forget-Me-Not™ EvaGreen® qPCR Master Mix is a ready-to-use high performance dye-based master mix for qPCR and DNA melt curve analysis. 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. For 2-color tracking, mix your template with Forget-Me-Not™ Template Buffer to see at a glance which wells have master mix or template.

  • Ready-to-use, just add primers and DNA template
  • Superior EvaGreen® Dye is more sensitive than SYBR® Green I and is compatible with HRM®
  • Prevent wasted samples and costly mistakes with Forget-Me-Not™ 2-color tracking
  • Formulated with Cheetah™ HotStart Taq for fast cycling protocols
  • Directly visualize amplification product fluorescence in a gel
  • No ROX or Separate ROX for use with any PCR instrument
Left: 1X Forget-Me-Not™ Master Mix. Right: 1X Forget-Me-Not™ Master Mix after addition of Forget-Me-Not™ Template Buffer.
Figure 1. Left: 1X Forget-Me-Not™ Master Mix. Right: 1X Forget-Me-Not™ Master Mix after addition of DNA template mixed with Forget-Me-Not™ Template Buffer.

Prevent Waste & Catch Mistakes

Catch pipetting mistakes and avoid wasting time, reagents, and your precious DNA samples. The 2-color tracking Forget-Me-Not™ EvaGreen® qPCR Master Mixes features a unique combination of a master mix containing a low concentration of blue dye and a DNA template buffer containing a high 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).  The Forget-Me-Not™ Template Buffer is optional if you do not require 2-color tracking. You can also use Forget-Me-Not™ EvaGreen® qPCR Master Mix (Low ROX or High ROX), which are premixed with ROX and allow single-color tracking.

Peace of Mind Plus PCR Performance

Forget-Me-Not™ qPCR Master Mix is formulated for fast cycling PCR parameters, but can also be used with regular cycling protocols. It performs as well as our Fast EvaGreen® Master Mix, and as well 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.

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 Qiagen’s 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).

EvaGreen® Dye

An environmentally friendly, non-mutagenic, and non-cytotoxic DNA-binding dye with features ideal for both qPCR and High Resolution Melt® (HRM) analysis. EvaGreen® Dye binds to dsDNA via a novel “release-on-demand” mechanism, which prevents dye transfer from amplicon to amplicon and permits the use of a relatively high dye concentration in qPCR without PCR inhibition. Learn more about EvaGreen® Dye for qPCR.

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.

ROX 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. Product #31041 is supplied without ROX, while product #31042 is supplied with a separate vial of ROX.  For master mixes with pre-mixed ROX, use Forget-Me-Not™ EvaGreen® qPCR Master Mix (Low ROX or High ROX)

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).
**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.

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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