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Page 1 of 4Fast EvaGreen qPCR Master Mix
Glowing Products for ScienceTM
3159 Corporate Place
Hayward, CA 94545
www.biotium.com
Product Information
Last updated: August 6, 2012
Fast EvaGreen® qPCR Master Mix
Catalog Number: 31003, 31003-1, 31003-2 or 31003-T
Unit Size: 200 reactions (cat # 31003), 500 reactions (cat # 31003-1), 5,000
reactions (cat# 31003-2), or 100 reactions (cat # 31003-T, trial size)
Components:
The product has two components: component A and component B. Component
A is 2X master mix containing EvaGreen® dye, dNTP, PCR buffer (including Tris
and MgCl2) and CheetahTM hot-start Taq polymerase. Component B is 10X Rox
reference, which may be required on certain ABI instruments (See protocol below).
Spectral Properties of EvaGreen® Dye:
The absorption and fluorescence emission spectra of DNA-bound EvaGreen®
dye are very similar to those of SYBR® Green I or FAM (see Page 4, Figure 1).
λabs/λem = 500/530 nm (DNA bound) λabs = 471 nm (without DNA)
Storage and Handling
Fast EvaGreen® Master Mix is shipped on blue ice and should be stored immediately upon arrival at -20°C. When stored under the recommended condition
and handled correctly, the kit should be stable for at least 6 months from the date
of receipt. Before use, thaw at room temperature and mix well by gentle vortexing.
After thawing, the master mix should be kept on ice before use. It can be refrozen
for storage.
Product Description
Fast EvaGreen® Master Mix is a ready-to-use hot-start mix for qPCR and
DNA melt curve analysis of PCR amplicons. It is formulated for qPCR using a fast
cycling protocol, but also can be used for qPCR using regular cycling protocols.
EvaGreen® dye is a unique DNA-binding dye with features ideal for both
qPCR and melt curve analysis. 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.
Fast EvaGreen® qPCR Master Mix contains CheetahTM Taq, our proprietary
chemically-modified hot-start DNA Polymerase. Unlike AmpliTaq Gold®, which
is also a chemically modified Taq but takes 10 minutes or longer to activate,
CheetahTM Taq is fully activated in 2 minutes with high activity recovery, making
it particularly suitable for fast PCR. Cheetah Taq is completely inactive at room
temperature and largely 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.
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.3 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.
An added benefit of 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 visualization of DNA bands following
electrophoresis.
References
1. Mao, et al. Characterization of EvaGreen Dye and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology 7, 76-91 (2007).
2. White, et al. Methylation-sensitive high-resolution melt-curve analysis of the
SNRPN gene as a diagnostic screen for Prader-Willi and Angelman Syndromes.
Clin. Chem. 53 (11), 1960-1962 (2007).
3. Ohta, et al. Ethidium bromide and SYBR Green I enhance the genotoxicity of
UV-irradiation and chemical mutagens in E. coli. Mutation Res. 492, 91-97 (2001).
Cat #
Component
31003A
(2X master mix)
99939
(10X ROX reference dye)
31003 2 X 1 mL 1 X 1 mL
31003-1 5 X 1 mL 1 X 1 mL
31003-2 50 X 1 mL 10 X 1 mL
31003-T 1 X 1 mL 1 X 1 mL
Page 2 of 4Fast EvaGreen qPCR Master Mix
PCR Protocols
General Considerations
1) qPCR instruments: For iCycler users, you do not need to add FAM to your PCR mix as
EvaGreen dye has a slight background fluorescence that provides adequate and stable
baseline level fluorescence. For Roche LightCycler users using glass capillaries for
reactions, you need to add BSA to your PCR reactions (~0.5 mg/mL final concentration).
BSA is not necessary if transparent plastic capillary tubes are used.
2) Instruments for melt curve analysis: Suitable instruments include Rotor-Gene 6000, ABI
7500 FAST and HR1™, 384-well LightScanner™ and Roche LightCycler 480. RotorGene 6000, ABI 7500 FAST and Roche LightCycler 480 are capable of performing both
qPCR and melt curve analysis. Follow the manufacturer’s instruction for data collection
and analysis.
3) Expected ∆R and ∆RN: When comparing signal strength among various commercial
qPCR master mixes, one needs to be mindful of the method used in the comparison.
Conventionally, ∆R is the fluorescence gain above the baseline. In general, 10 µL of 1X
Fast EvaGreen® reaction generates higher ∆R than 50 µL 1X PowerSYBR from ABI or
1X SYBR GreenER from Invitrogen. ∆Rn is defined as ∆R divided by the signal in the
ROX channel. Therefore, a higher concentration of ROX will generate smaller ∆Rn. ∆Rn
will also become smaller when ROX is excited at its maximal as in the case of ABI 7500,
iCycler IQ, MJ opticon, MJ Chromo4, MX3000, and MX4000. Accordingly, the lower
ROX concentration used in some commercial SYBR Green master mixes will produce a
higher ∆Rn.
4) Expected kinetic curve: Based on our comparative studies, amplification curves of Fast
EvaGreen® Master Mix generally are more robust than other commercial master mixes
formulated using SYBR Green I. Because of SYBR’s inhibitory effect, SYBR-based
master mixes may tend to stall amplification 5-7 cycles after the signal reaches the Ct
threshold. In contrast, reactions in Fast EvaGreen® Master Mix can continue to amplify
for as many as 50 cycles.
5) Expected Ct value: Under similar conditions, Ct values generated by EvaGreen and
SYBR Green I may differ from each other by +1 or –1.
6) Amplicon length: To maximize amplification efficiency with Fast EvaGreen master mix,
the optimal amplicon length is 50-200 bp. For longer amplicons you may need to extend
the elongation time.
7) Gel electrophoresis analysis of PCR product: To analyze your PCR product by gel
electrophoresis using the EvaGreen® dye in the master mix as a prestain, simply
add DNA loading buffer your PCR reaction solution, load on a gel, and conduct
electrophoresis as usual. No additional DNA-binding dye needs to be added to either the
loading buffer or the gel. Gel visualization can be carried out using a 254 nm UV box,
or a gel imager or Dark Reader using a SYBR Green filter. Alternatively, the gel may be
imaged using a 488 nm laser-based gel scanner.
PCR Reaction Setup
Pipet reaction components into each well according to the table below:
Reaction component Amount required per 20 uL reaction Final concentration
2X Fast EvaGreen
Master Mix 10 uL 1X
Primers x uL each 0.1-0.5 uM each
Template x uLSee Notes #1 & #2 See Note #3
ROX Optional See Note #4 and Table 1
H2O Add to 20 uL
PCR Instrument Recommended Rox Concentration Amount of 10X ROX per 20 uL reaction
BioRad: iCycler, MyiQ, MiQ 2, iQ 5, CFX-96, CFX-384, MJ Opticon, Option2,
Chromo4, MiniOpticon
Qiagen: Rotor-Gene Q, Rotor-Gene3000, Rotor-Gene 6000
Eppendorf: Mastercycler realplex
Illumina: Eco RealTime PCR System
Cepheid: SmartCyler
Roche: LightCycler 480, LightCycler 2.0
No ROX None
ABI: 7500, 7500 Fast, ViiA 7
Stratagene: MX4000P, MX3000P, MX3005P
Low ROX
0.05-0.1X final
Dilute 10X ROX 1:10 with dH2O to obtain 1X ROX;
add 1 to 2 uL of 1X ROX per 20 uL reaction
ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne plus High ROX1X final 2 uL of 10X ROX per 20 uL reaction
Table 1. Recommended ROX Concentration for PCR Instruments
Notes:
1) cDNA templates: Fast EvaGreen® master mix is suitable for mRNA quantitation if a twostep procedure is followed. The first step involves converting the mRNA to cDNA by reverse
transcription (components not provided). A portion of the synthesized cDNA can then be
quantitated by using Fast EvaGreen kit in the second step. 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. We recommend cDNA synthesis kits from Quanta or Invitrogen.
For accurate quantitation of transcript levels, a no-RT control is recommended to check for
possible genomic DNA contamination.
2) One-step RT-qPCR can also be applied for mRNA quantitation. Primer sets 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 oC, run both
RT step and extension step at 55 oC. For accurate quantitation of transcript levels, a no-RT
control is recommended to check for possible genomic DNA contamination.
3) Template concentration: The optimal amount of template DNA varies by application.
Recommended amounts of genomic DNA template per reaction typically range from 50 pg
to 50 ng per reaction. Recommended amounts of cDNA typically range from 50 fg to 50 pg,
based on the amount of input RNA in the RT reaction.
4) ROX reference dye: For certain instruments, ROX is necessary for accurate Ct
determination from well to well. Refer to Table 1 for the recommended ROX concentration for
your instrument. ROX may add noise to melt curve analysis, which could be mistaken for real
peaks. Thus, in case of unexpected peaks, un-check “ROX” in the “Passive Reference Dye”
box in the software so that data is not collected from the ROX fluorescence channel, then
re-analyze the data.
Page 3 of 4Fast EvaGreen qPCR Master Mix
Cycling Protocols
You may choose one of the following three protocols, depending on the nature of your amplicon and instrument capability.
A. Two-step fast cycling protocol
This cycling protocol should be applicable to most amplifications where the primer Tm’s are designed to be 60 oC. Melt curves may be
performed by following instructions provided for your instrument.
Note:
5) Denaturation time: The holding time for denaturation can be lower than 5 seconds, including as low as 0 second, if
you have a relatively short amplicon. When the denaturation time is set to “0” in the program, it merely means that the
temperature is ramped up to 96 oC and then immediately ramped down with no stay. Setting the time to 5 s will ensures
a more robust denaturation for relatively long or high GC amplicons. Instruments with fast ramping capability further
add reliability to amplicon denaturation.
B. Three-step fast cycling protocol
This cycling protocol can be used if you would like to have the extension step to be performed at a higher temperature than the annealing step. For example, if you have relatively long primers that tend to anneal non-specifically, carrying out the extension step at a higher
temperature can reduce nonspecific amplification. Melt curves may be performed by following instructions provided for your instrument.
Cycling Step Temperature Holding Time Number of Cycles
Enzyme activation 95 oC 2 min 1
Denaturation
Annealing & Extension
95 oC
60 oC
5 s (See Note #5)
30 s
45
Notes:
6) Annealing temperature: The annealing temperature should be set at your primer Tm, which should generally be
50-60 oC for optimal result. However, whenever possible, primer Tm (and thus extension temperature) should be
designed closer to 60 oC (but still within 50-60 oC range) to minimize the gap between annealing and denaturation
temperatures. This way, the temperature ramping will take less time, which in turn facilitates amplification.
7) Extension temperature: Extension at 72 oC is usually more efficient for most amplicons. However, for AT-rich amplicons (>70% AT) or amplicons that have an AT-rich patch, extension at 60 oC usually gives better results.
Cycling Step Temperature Holding Time Number of Cycles
Enzyme activation 95 oC 2 min 1
Denaturation
Annealing
Extension
95 oC
50-60 oC (See Note #6)
72 oC (See Note #7)
5 s
5 s
25 s
45
C. Universal cycling protocol
This cycling protocol can be used on nearly all qPCR instruments. The protocol also may be useful for targets that are relatively difficult
to amplify under fast cycling conditions.
Cycling Step Temperature Holding Time Number of Cycles
Enzyme activation 95 oC 2 min
Denaturation
Annealing & Extension
95 oC
60 oC
15 s
60 s
45
Page 4 of 4Fast EvaGreen qPCR Master Mix
Appendix. EvaGreen Dye® Characteristics
The following figures provide additional information on EvaGreen® dye in
regard to its fluorescence spectra, stability and cell membrane permeability.
Safety of EvaGreen Dye
Ames testing performed by an independent lab, Litron Laboratories (Rochester, NY), showed that EvaGreen® dye is nonmutagenic as well as noncytotoxic.
EvaGreen® dye appears to be completely cell membrane-impermeable (Figure 3),
which may be a key factor responsible for the observed low toxicity. On the other
hand, SYBR Green I is known to be a powerful mutation enhancer, possibly by
inhibiting the natural DNA repairing mechanism in cells (Ohta, et el. Mutat. Res.
492, 91(2001)). The toxicity of SYBR Green I may be associated with its ability to
enter cells rapidly (Figure 3).
For more information on the Ames test results, you may download the
EvaGreen® safety report from www.biotium.com.
Wavelength (nm)
E
xc ita
tio
n E
m is si on Spectral Characteristics
Figure 1. Excitation (left) and emission (right) spectra of EvaGreen® dye bound to dsDNA in
pH 7.3 PBS buffer. Also see ref. 1.
Stability Comparison of EvaGreen Dye and SYBR Green I
Figure 3. HeLa cells were incubated with SYBR Green I (1.2 µM) or EvaGreen dye (1.2 µM) at
37 oC. Photographs were taken following incubation for 5 min (panel A) and 30 min (panel B).
SYBR Green I stained cells rapidly while EvaGreen appeared to be membrane impermeable.
Comparison of Cell Membrane Permeability
between EvaGreen Dye and SYBR Green I
A) 5 min. incubation
B) 30 min. incubation
SYBR Green I, 1.2 µM EvaGreen dye, 1.2 µM
SYBR Green I, 1.2 µM EvaGreen dye, 1.2 µM
Biotium products are high-quality reagents and materials intended for research purposes only. EvaGreen® dye and applications are covered under patent US patent nos. 7,803,943 and 7,776,567 and pending international patents.
Our products are not available for resale or other commercial uses without a specific agreement from Biotium, Inc. We welcome inquiries about licensing the use of our dyes, trademarks or technologies. Please submit inquiries by
e-mail to btinfo@biotium.com. EvaGreen® is a registered trademark of Biotium, Inc.
NOTICE TO PURCHASER: LIMITED LICENSE
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, non-transferable immunity from
suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim and no right to perform commercial services of any kind, including without
limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents
require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights are conveyed expressly, by
implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
Practicing real-time PCR may require additional licensing from Roche or Applied Biosystems, Inc. Practicing HRM may require a licence from Idaho Technologies. SYBR® is a registered trademark of Invitrogen, Inc.
Related Products:
EvaGreen dye, 20X in H2O, cat# 31000
Cheetah hotstart Taq DNA polymerase, cat# 29050
Fast Plus EvaGreen Master Mix, no ROX, cat# 31020
Fast Plus EvaGreen Master Mix, low ROX, cat# 31014
Fast Plus EvaGreen Master Mix, high ROX, cat# 31015
PMA for selective detection of live pathogens by PCR, cat# 40013
GelRed nucleic acid gel stain, 10,000X in H2O, cat# 41003
GelGreen nucleic acid gel stain, 10,000X in H2O, cat# 41005
Figure 2. A solution of EvaGreenTM dye or SYBR Green I each at 1.2 µM in pH 9 Tris buffer
was incubated at 99 oC. The absorption spectrum of each solution was followed over a period
of 3 hours. ROX was added as a stable reference.

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