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).
For most qPCR applications, we recommend using EvaGreen® Dye, 20X in water (31000), or one of our 2X EvaGreen® qPCR Master Mixes. EvaGreen® Dye, 2000X in DMSO (31019) is offered for specialized applications requiring a concentrated dye solution.
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
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.
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)
*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.