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

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.

Note: We also offer 2X Master Mix (99801-10ML) for standalone purchase for customers who do not wish to use template buffer.

• 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

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.

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
Low ROX (~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 Opus, 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.