The Technologies

Key Features√√√√√ Superior sensitivity and wide dynamic range

√√√√√ Suitable for both qPCR and HRM

√√√√√ Compatible with both fast and regular cyclingprotocols

√√√√√ Novel chemically-modified hotstart Taqrequiring only 2 minutes to activate

√√√√√ Suitable for TaqMan assay as well, enablingmulti-detections by both EvaGreen dye andoligo probes, followed by melt curve analysis

Fast EvaGreen qPCR master mix is a PCR reagentsuitable for both qPCR and high resolution meltcurve (HRM) analysis. The reagent system uses twoof our breakthrough PCR technologies, EvaGreenqPCR dye and Cheetah Taq hotstart DNApolymerase, to deliver superior results comparedto other commercial master mixes.

EvaGreen Dye: EvaGreen dye is the nextgeneration DNA-binding dye ideal for both real-time PCR detection and HRM. The dye selectivelybinds to dsDNA via a novel “release-on-demand”mechanism that ensures low PCR inhibition andpermits HRM application at below saturation dyeconcentration. Because the dye is spectrallysimilar to FAM or SYBR Green I, it is compatiblewith all commercial qPCR instruments. Moreover,EvaGreen dye is nonmutagenic and extremelystable.

Cheetah Taq: Cheetah Taq is a new, chemicallymodified hotstart Taq DNA polymerase superior toAmpliTaq Gold. Prepared from Taq and aproprietary Hot-off chemical modifier,Cheetah Taq can be activated in as little as twominutes under the standard hotstart condition(i.e., 94 °C in pH 8-9 Tris) with high activityrecovery. Additionally, Cheetah Taq is morestable at -20 °C or 4 °C than AmpliTaq Gold,ensuring that the master mix maintain its peakperformance following storage at lowtemperature. Cheetah Taq is more advantageousthan any antibody-based hotstart polymerase,such as Platinum Taq, because it is completelyinactive at room temperature and alsointrinsically less susceptible to DNAcontamination.

Figure 1. Comparison among Fast EvaGreen master mix from Biotium and two fast SYBR Green mastermixes from two leading companies (company A and company Q) in the amplification of ATPG fragment (a104-bp fragment encoding ATP gene) of human genomic DNA.. Reactions were carried out on the same plateusing ABI 7900 Fast on DNA inputs of 200 ng, 20 ng, 2 ng, 200 pg, 20 pg and 0 pg, respectively. Thepassive reference dye, ROX, was adjusted for each master mix so that the final ∆Rn faithfully reflects thefluorescence change of each master mix. To accomodate the relatively slow activation of company Qmaster mix, the entire plate was activated at 95 ºC for 5 minutes. The cycling was carried out between 96 ºC (5 s) and 60 ºC (30 s). Data set from the 200 ng DNA input are highlighted in the plot for easy distinguishingfrom the remaining amplification curves. The inset is an enlarged view of the area near the baseline for betterviewing the curve patterns of the much weaker signals of the two SYBR-based master mixes.

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Amplification of Human Genomic DNA

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Company A 200 ngCompany A 20 ngCompany A 2 ngCompany A 200 pgCompany A 20 pgCompany A NTCCompany Q 200 ngCompany Q 20 ngCompany Q 2 ngCompany Q 200 pgCompany Q 20 pgCompany Q NTCFast EvaGreen 200 ngFast EvaGreen 20 ngFast EvaGreen 2 ngFast EvaGreen 200 pgFast EvaGreen 20 pgFast EvaGreen NTC

Suitable for both qPCR and HRM with unrivaled performance

Fast EvaGreen® qPCR Master Mix

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Fast EvaGreen master mix has been tested on a

variety of templates, including templates that are

short (<100 bp), long (>700bp), AT-rich, GC-rich or

of genomic origin. The results show, Fast

EvaGreen master mix is highly robust and

superior to SYBR-based master mixes from other

suppliers. Figures 1 and 2 compare Fast

EvaGreen master mix from Biotium with SYBR-

based fast qPCR master mixes from two leading

companies (company A and company Q) in the

amplifications of human genomic DNA and

human brain cDNA, respectively. The results

demonstrate that Fast EvaGreen Master Mix is

significantly more sensitive and gives a wider

linear detection range. The advantage of Fast

EvaGreen master mix is especially striking for

the more challenging amplification of human

genomic DNA (Figure 1); the EvaGreen master

mix gives early Ct values and a wide 5-log linear

detection range while the master mix from

company Q and, in particular, the master mix from

company A suffer from significant Ct delay and

narrow linear detection range (as suggested by

the irregular spacings between the curves).

Results on melt curve analysis mirror those on

the qPCR for the three master mixes as shown in

Fast EvaGreen® Master Mix

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The Performance

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Fast EvaGreen

Figure 3. Comparison of Fast EvaGreen master mix and fast qPCR master mixesfrom company A and company Q in melt curve analysis. Analyses were carried outfollowing the amplification of 200 ng of human genomic DNA using each of the threemaster mixes, respectively, as detailed in Figure 1 legend.

Figure 4. Comparison of Fast EvaGreen master mix and fast qPCR master mixes from company Aand company Q in melt curve analysis. Analyses were carried out following the amplification of 400pg of human brain cDNA using each of the three master mixes, respectively, as detailed in Figure 2legend.

Figures 3 and 4; melt curves with Fast EvaGreen master

mix are far stronger than those with the two other SYBR-

based fast master mixes.

Figure 2. Comparison among Fast EvaGreen master mix from Biotium and two fast SYBR Green mastermixes from two leading companies (company A and company Q) in the amplification of a B2M fragment (a71-bp fragment encoding B2M transcript) of human brain cDNA.. Reactions were carried out on the sameplate using ABI 7900 Fast on DNA inputs of 400 pg, 40 pg, 4 pg, 0.4 pg, 0.04 pg and 0 pg, respectively.The passive reference dye, ROX, was adjusted for each master mix so that the final ∆Rn faithfully reflectsthe fluorescence change of each master mix. To accomodate the relatively slow activation of company Qmaster mix, the entire plate was activated at 95 ºC for 5 minutes. The cycling protocol was: 96 ºC for 5 s,60 ºC for 5 s and 72 ºC for 25 s. The inset is an enlarged view of the area near the baseline for betterviewing the curve patterns of the much weaker signals of the two SYBR-based master mixes.

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Company A 400 pg

Company A 40 pg

Company A 4 pg

Company A 0.4 pg

Company A 0.04 pg

Company A NTC

Company Q 400 pg

Company Q 40 pg

Company Q 4 pg

Company Q 0.4 pg

Company Q 0.04 pg

Company Q NTC

Fast EvaGreen 400 pg

Fast EvaGreen 40 pg

Fast EvaGreen 4 pg

Fast EvaGreen 0.4 pg

Fast EvaGreen 0.04 pg

Fast EvaGreen NTC

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Cycle N u mb er

Amplification of Human cDNA

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Fast EvaGreen

both applications. SinceEvaGreen dye has very lowPCR inhibition because of itsnovel "release-on-demand"DNA-binding mechanism, arelatively high dyeconcentration can be used inqPCR for maximal signalstrength. This same dyeconcentration is also ideal forHRM, resulting in a singlemaster mix optimal for bothapplications.Another unique application ofFast EvaGreen master mix isqPCR detection by bothEvaGreen dye and one ormore oligo probes (e.g.,TaqMan probes or our AllGloprobes), all in the samereaction well. In thistechnique, EvaGreen dyemonitors total DNA productionin the green channel while theoligo probes detectamplifications of individualtargets in the red channels.Moreover, post PCR melt curveanalysis can be performed toconfirm the identities andnumber of products amplified.Figures 5-7 illustrate theutilities of Fast EvaGreenmaster mix in these additional

In addition to its qPCRapplication, Fast EvaGreenmaster mix can also be usedfor high resolution melt(HRM) analysis and oligoprobe-based qPCR assay.HRM is a recently developedDNA analysis techniquecapable of detecting singlemutations. It is gaining rapidpopularity due to itssimplicity and relatively lowcost. An essentialrequirement for successfulHRM is that the concentrationof the DNA-binding dye beabove or well above theoptimal dye concentrationused in qPCR for SYBR GreenI and other similar DNAbinding dyes. As a result, forthese dyes it is relativelydifficult to formulate a singlemaster mix optimal for bothapplications. Although someof the commercial mastermixes using SYBR or otherdyes are promoted to beapplicable for both qPCR andHRM, their performanceusually suffers because acompromise has to be madein selecting a dyeconcentration sufficient for

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The Versatility

Figure 4. Amplifications of B2M gene fragment and its two mutants mutant 1 and mutant 2, respectively,using Fast EvaGreen master mix containing both EvaGreen dye and a NEP-labeled AllGloTM probe (aTaqMan-like fluorogenic oligo probe). Mutant 1 has a single point mutation outside the the probehybridization region; Mutant 2 has a single point mutation in the middle of the probe. In eachamplification, EvaGreen signal (dashed lines) is recorded in the green (FAM) channel and the probesignal (solid lines) in the red (Cy5) channel. The wild type (green) and mutant 1(red) each producedpositive signals in both FAM (dashed)and Cy5 (solid) channels. Mutant 2 (blue) produced a positve signalonly in the FAM channel but essentially no positive signal in the Cy5 channel due to failure in probehybridization.

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Figure 5. HRM analyses on products generated in Figure 4. Data are plotted in the form ofnormalized fluorescence vs. temperature (top plot) or fluorescence difference vs.temperature (bottom plot). In both plots, only one representative curve for each genotype ispresented. HRM successfully resolved all three genotypes.

Fast EvaGreen master mix is amongthe most competatively priced qPCRmaster mixes on the market. Coupledwith its unmatched performance, Fast

EvaGreen master mix offers you thebest value. We can afford to offeryou the competitive price because weare the original inventors of thetwo key technologies in the product:EvaGreen qPCR dye and Cheetahhotstart Taq DNA polymerase.

The Value

applications.

Fast EvaGreen master mix is available in thesepackaging sizes:

Cat# 31003, 200 reactions Cat# 31003-1, 500 reactions Cat# 31003-2, 5,000 reactions

Reaction volume is 20 µµµµµL/well. ROX referencedye is provided in separate vials. A detailedprotocol is included with each product and canalso be downloaded at Biotium website.

Biotium, Inc., 3423 investment Blvd., Hayward, CA 94545

Tel: 1-800 304 5357 Fax: 510 265 1352 btinfo@biotium.com

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#Cat. Product Name Unit Size Application

31000EvaGreen dye, 20Xin H2O

L5X1 mHEvaGreen dye 20X concentrated in ultra pure 2 dO is specifically formulate

for qPCR application. Please ask for licensing opportunity.

29050Cheetah MT thotstaTaq DNApolymerase

200 rxn

gCheetah Taq is a hotstart DNA polymerase prepared by covalently attachina proprietary chemical modifier to Taq. Taking only 2 minutes to activateand with its excellent stability during storage, this chemically modifiedhotstart Taq is superior to AmpliTaq Gold. It is also more advantageous thanantibody-based hotstart enzymes due to intrinsically low DNA contaminationand complete lack of enzyme activity at room temperature. Variouspackaging sizes are available. Please ask for licensing opportunity.

40013PMA MT ADNmodification agent

1 mg

ePMA is a cell membrane-impermeable DNA modification dye that can bused to selectively covalently modify DNA from dead cells, rendering themodified DNA unamplifiable. Subsequently, DNA from viable cells can bequantified by qPCR. Thus, PMA can be used in selective detection of viablepathogens in the presence of dead pathogens by qPCR.

41003GelRed MT cnucleiacid gel stain,10,000X in H2O

L0.5 m

eGelRed is the perfect replacement for the highly toxic ethedium bromid(EB) for nucleic acid gel staining. It is far more senstive than EB but isnonmutagenic, noncytotoxic and safe to dispose in the drain. The dye isspectrally similar to EB, so it is completely compatible with existinginstruments.

41005GelGreen MT cnucleiacid gel stain,10,000X in H2O

L0.5 mrSimilar to GelRed, GelGreen is a nucleic acid gel stain with superio

senstivity and excellent safety profile. GelGreen is ideal for visible lightexcitation, which avoids DNA damage by UV light.

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