Fast-Plus EvaGreen® qPCR Master Mix
Superior Performance with an Environmentally Safe Formulation
Fast-Plus EvaGreen® qPCR master mix is a ready-to-use hot-start mix for nucleic acid quantitation and melt curve analysis of PCR amplicons. Formulated using our environmentally safe EvaGreen® dye and fast-activating chemically-modified hotstart enzyme, Cheetah™ Taq, Biotium's PCR master mix delivers superior performance even with the most challenging samples tested. The master mix is offered in three formats containing different amounts of ROX reference dye to best suit for a variety of PCR instruments.
A critical component of the master mix is EvaGreen® dye, a unique DNA-binding dye with features ideal for both qPCR and high-resolution melt analysis (HRM).* EvaGreen® dye binds to dsDNA via a novel “release-on-demand” mechanism, which permits saturation of dye concentration in qPCR without PCR inhibition. Another important feature of EvaGreen® dye is its safety. Very few PCR dyes have been thoroughly studied for their safety despite the increasing use of PCR in research and diagnostics and the fact that DNA-binding dyes are inherently dangerous due to their potential to cause mutation. Thus, handling and disposal of PCR master mixes can be a health and environmental issue. Indeed, SYBR® Green I, a popular PCR dye, is found to be even more environmentally toxic than ethidium bromide, one of the best known mutagen (Ohta, et al. Mutation Research, 492, 91-97(2001)). SYBR® Green I has been suggested to interfere with the natural DNA-repair mechanism in cells and as a result it potentiates genotoxicity of chemicals as well as DNA damage by UV light. With this in mind, Biotium’s scientists designed EvaGreen® dye such that it cannot cross cell membranes, thus preventing the dye from coming into contact with genomic DNA in live cells. Independent labs have confirmed that EvaGreen® dye is nonmutagenic, noncytotoxic and safe to aquatic life for direct disposal in the drain. (Visit Biotium website for a full EvaGreen® dye safety report).
- Formulated using the first and only safe PCR dye: EvaGreen® dye, which has passed California environmental regulation (CCR Title 22) for disposal down the drain
- Unique "release-on-demand" DNA binding mechanism of EvaGreen dye enables superior PCR and melt curve analysis results*
- Novel chemically-modified hotstart Taq, Cheetah™ Taq, requiring only 2 minutes to activate
- Compatible with both fast and regular cycling protocols
- EvaGreen dye in the master mix can also serve as a gel stain — Analyze your PCR product by gel electrophoresis without the need to add another gel stain
* Practicing HRM may require a license from Idaho Technologies, Inc.
Another important component of the master mix is Cheetah™ Taq, our proprietary chemically-modified hot-start DNA polymerase. Unlike AmpliTaq Gold®, which takes 10 minutes or longer to activate, Cheetah™ Taq is fully recovered in 2 minutes with high activity, making it particularly suitable for fast PCR. Cheetah™ Taq is completely inactive at room temperature and mostly free of DNA contamination. This makes Cheetah™ Taq superior to any antibody-based hotstart Taq, which is typically not completely inactive at room temperature and is prone to DNA contamination due to the nature of antibody production.
The master mix is suitable for two-step mRNA quantitation by first converting mRNA to cDNA via reverse transcription (components not provided), followed by quantitating a portion of the cDNA using the master mix. To ensure optimal amplification efficiency, the aliquot of the cDNA sample to be amplified should not exceed 10% of the volume of the PCR reaction. For accurate quantitation of mRNA level, a none-RT control is recommended to check for the possibility of genomic DNA contamination.
One-step RT-qPCR can also be applied for mRNA quantitation. Primer set must be well characterized to ensure no primer-dimer formation. We recommend that you titrate the amount of reverse transcriptase and the duration of the RT step. Heat-resistant reverse transcriptases that have been tested to be compatible include those from Agilent, Fermentas, Lucigen and Life Technologies. If possible, design primers to have Tm at 55°C, run both RT step and extension step at 55°C. For accurate quantitation of mRNA level, a none-RT control is recommended to check for the possibility of genomic DNA contamination.
Another benefit of the Fast-Plus EvaGreen® 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 prestain, permitting direct and immediate visualization of DNA bands following electrophoresis.
Cell impermeability of EvaGreen® dye makes it the only PCR dye to date that is non-mutagenic, non-cytotoxic and safe to aquatic life
|A) 5 min. incubation||B) 30 min. incubation||C) 30 min. incubation, long photographing exposure time||Figure 2. Comparison of cell membrane permeability between EvaGreen® dye and SYBR® Green I. HeLa cells were incubated with SYBR® Green I (1.2 µM) or EvaGreen® dye (1.2 µM) at 37 °C. Photographs were taken following incubation for 5 and 30 minutes. SYBR® Green I entered cells rapidly while EvaGreen® dye appeared membrane-impermeable as evident from the absence of cell nuclear staining. Image taken with long photo-exposure time revealed that EvaGreen® dye only associated with cell membranes. SYBR® Green I has been suggested to interfere with the DNA repair mechanism in living cells, a rationale used to explain the observation that the dye is even more environmentally toxic than ethidium bromide (Ohta, et al. Mutation Research, 492, 91-97(2001)). In contrast, EvaGreen® dye has been confirmed to be non-mutagenic and non-cytotoxic. See full EvaGreen dye safety report at Biotium website.|
|SYBR Green I|
Table 1. Fast Plus EvaGreen® Master Mix selection guide
|Product Group||Cat #||Packaging Size||Component||PCR Instrument|
Master Mix (no ROX)
|31020||200 rxn (2 X 1 mL)||EvaGreen® dye, dNTP, buffer composition (including Tris and MgCl2) and CheetahTM hot-start Taq polymerase. Optimized for most of the non-ABI instruments which do NOT require ROX reference dye.||BioRad: iCycler, MyiQ, MiQ 2, iQ 5,
CFX-96, CFX-384, MJ Opticon, Option2, Chromo4, MiniOpticon
Qiagen: Roto-Gene Q, Roto-Gene3000, Roto-Gene 6000
Eppendorf: Mastercycler realplex
Illumina: Eco RealTime PCR System
Roche: LIghtCycler 480, LightCycler 2.0
|31020-1||500 rxn (5 X 1 mL)|
|31020-2||5,000 rxn (50 X 1 mL)|
Master Mix with Low ROX
|31014||200 rxn (2 X 1 mL)||EvaGreen® dye, dNTP, buffer composition (including Tris and MgCl2), CheetahTM hot-start Taq polymerase and low concentration of ROX reference dye.||ABI: 7500, 7500 Fast
Stratagene: MX4000P, MX3000P,
|31014-1||500 rxn (5 X 1 mL)|
|31014-2||5,000 rxn (50 X 1 mL)|
Master Mix with High ROX
|31015||200 rxn (2 X 1 mL)||EvaGreen® dye, dNTP, buffer composition (including Tris and MgCl2), CheetahTM hot-start Taq polymerase and high concentration of ROX reference dye.||ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne plus|
|31015-1||500 rxn (5 X 1 mL)|
|31015-2||5,000 rxn (50 X 1 mL)|
Table 2.Ordering Information
|Unit Size||Unit Price|
|** Price is for US enduser only. International price may vary. Please contact your local distributors for your price.|