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Applied Biosystems PowerTrack SYBR Green Master MixFebruary 4, 2020The world leader in serving science
Table of ContentsTopic2SlidenumberDifferences between TaqMan and SYBR Green Real-Time PCR (otherwise known asquantitative polymerase chain reaction – qPCR) chemistries3Recommended Real-Time PCR master mix by application4Requirements for a high quality SYBR Green Experiment5PowerTrack SYBR Green product overview / key benefits / how does it work / keyapplications6-8Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines(MIQE), Researcher Challenges and benefits, ROX dye9 -11Performance data12 - 15Product configurations, Technical specs and for more information16 - 18Appendix (experimental conditions and additional data)19 - 23
qPCR Technologies: SYBR Green chemistry and TaqMan AssaysSYBR Green ChemistryTaqMan Assays Detection based on SYBR Green, a nonspecific DNA binding dye Specificity based on 2 hybridization events:PCR 3’5’ Detection based on 5’ nuclease activity Specificity based on 3 hybridization events:two PCR primers & a probe and cleavage ofthe imer3’5’ Customer designed primers Accuracy & sensitivity for highest performance Plenty of sample Multiplexing up to 4 targets per well Time to optimize primers Low sample input Only 1 target per well Convenient, Predesigned TaqMan assays Low priced (but takes time) Higher price (but saves time)
Recommended master mix by applicationChemistryTaqMan SYBR ApplicationStart with.Gene ExpressionTaqMan Fast Advanced MasterMixfor best performance 2-stepTaqMan Fast Virus Master Mixfor 1-step virus detection and gene expressionGenotyping (seebelow)Pre-ampTaqMan Pre-AMP Master Mixfor customers with limited sample (e.g. FFPE)Gene ExpressionPowerTrack SYBR Green Master MixGenotyping and CopyNumber Variation (CNV)TaqPath ProAmp Master MixTaqPath ProAmp Multiplex Master MixTaqPath (Clinical-GradeTaqMan)General purpose reagent.For laboratory use.Molecular DiagnosticsDevelopmentTaqPath qPCR Master Mix(DNA detection or 2-step Gene Expression)TaqPath 1-step RT-qPCR Master MixTaqPath 1-step Multiplex Master Mix(RNA detection)
Requirements for a high quality SYBR Green assay experiment1 Verify specificity (design/create primers)2 Test dynamic range and efficiency3 Achieve high reproducibility and fold discrimination4 Determine sensitivity5 Confirm performance robustness across variety of analytes675 Compatibility with workflow Instruments performance Reaction handling Ensure no contamination
PowerTrack SYBR Green Master Mix OverviewEase of use and Flexibility at a CompetitivePriceFor SYBR Green gene expression, performanceseekers looking for broad dynamic range andminimize pipetting errors, PowerTrack SYBRGreen Master Mix offers TRACKING dye to reduce pipetting errors BROAD primer Tm and concentration allowsflexibility and minimal optimizationSUPERIOR specificity & sensitivity 6 TIGHT reproducibility UNIVERSAL instrument compatibilityReproducibility -- Specificity -- Sensitivity -- Compatibility -- Price
How does it work?YELLOW sample bufferPipette BLUE Master Mix into the tubes/platesBLUE Master MixAdd YELLOW sample buffer to your cDNA/DNAsample (optional)Indicates that sample has been added toMaster Mix7
PowerTrack SYBR Green Target ApplicationsGene expression Relative and absolute quantification of gene expression levels using SYBRValidation Validation of subset of microarray gene targets Validation of subset of NGS gene targetsScreening Quick screening of cell lines for targets8
Specificity is critical to SYBR Green Reactions Specific amplification is essential for valid data and part of the MinimumInformation for Publication of Quantitative Real-Time PCR ExperimentsGuidelines (MIQE) Non-specificity can be observed as multiple MELT peaks following adissociation curve analysis If non-specific amplification occurs, researcherscan try to optimize reaction componentconcentrations or re-design primers. Of researchers using SYBR chemistry 78% verify specificity of qPCR primers 42% perform primer optimization at least 25% of the time 15% redesign primers at least 25% of the timeNon-specificity compromises data quality and costs researchers extra time andmoney to address9
Researcher challenges and benefitsPowerTrack SYBR Green Master MixFor gene expression analysis customers who need broad dynamic range and to minimize pipetting mistakeswithout compromising sensitivity and specificity, PowerTrack SYBR Green Master Mix contains a tracking dyeallowing users to know where they have pipetted:ChallengeBenefitHow25% of poor real-time PCR datacomes from pipetting errorsHelps reduce pipetting errorsTwo-color tracking dye indicateswhere pipetting has occurredNeed for specific target amplification Accurate and specificSpecific and tight reproducibility inCt’s over a broad dynamic rangeDifferent targets have varyingannealing temperaturesFlexibleBroad primer Tm and primerconcentration compatibility allowsqPCR reaction set up flexibility withminimal optimizationThe need for high-yield of reversetranscriptaseCompatible with preferred RT kitDesigned to be used withSuperScript IV VILO Master Mix10
Why ROX dye matters ROX dye is an inert reference dye used to normalize fluorescence across all samples. It removesfluorescent variations, such as those caused by bubbles from the data For example if you have a bubble it can cause a difference in fluorescence, where ROX dyecan normalize for this Unlike other suppliers, Applied Biosystems ROX dye is universal, therefore eliminating theneed for ‘low’ or ‘high’ ROX dye versions to accommodate different instruments It will help improve your precision which can improve your data or reduce the number ofreplicates required11
Melt curve data shows PowerTrack SYBR Green Master Mix has higher specificitycompared to other suppliers shown by a single peakCompetitor Q1Competitor BRCompetitor BL PowerTrackamplifiesspecificproduct asshown bysingle peak vs.other suppliersmixes showingprimer dimersand secondarypeak. 2nd peak isindicative ofnon-specificamplification inother suppliersmixes.SNF8ARL1PGK1PowerTrack12 Thermal cycling performed using 60ºC primer annealing Tm with UHR cDNA in fast cycling mode.10µl reactions run in quadruplicate on QuantStudio5 real time PCR instrument, 384 well blockPGK1 – High expressor; ARL1 – Medium expressor; SNF 8 – Low expressor
PowerTrack SYBR Green Master Mix powers through traditionally difficult targetsPGK1 13ARL1IGF2TCF25Ten-fold dilutions of UHR cDNA ranging from 100ng to 100fg were run on Applied Biosystems QuantStudio5 Real time PCR instrument with384 well block.10 µl reactions run in quadruplicate using 60ºC annealing primer Tm. PGK1 – High Expressor, ARL1, IGF2 and TCF25 - Medium Expressor
PGK1; Invitrogen SuperScript IV VILO Master Mix reverse transcriptionPowerTrack SYBR Green Master MixcDNA inputcDNA inputQiagen QuantiNova SYBR GreenBio-Rad SsoAdvanced Universal SYBR Green SupermixCt MeanCt Std. Dev.1. 100 ng/uL0.000.002. 10 ng/uL16.220.073. 1 ng/uL19.050.064. 100 pg/uL22.240.085. 10 pg/uL25.650.116. 1 pg/uL29.160.167. 100 fg/uL32.690.688.NTC40.000.00Efficiency (%): 100.34Slope: 3.314R2: 1.00Ct MeanCt Std. Dev.1. 100 ng/uL0.000.002. 10 ng/uL15.790.043. 1 ng/uL18.830.054. 100 pg/uL22.170.055. 10 pg/uL25.700.076. 1 pg/uL29.410.167. 100 fg/uL33.801.478.NTC40.000.00Efficiency (%): 90.20Slope: 3.582R2: 0.99cDNA inputCt MeanCt Std. Dev.1. 100 ng/uL0.000.002. 10 ng/uL15.500.083. 1 ng/uL18.380.044. 100 pg/uL21.680.055. 10 pg/uL25.150.096. 1 pg/uL28.600.257. 100 fg/uL32.430.158.NTC34.310.00Efficiency (%): 97.64Slope: 3.380R2: dCt2.873.303.483.453.83Other suppliersshow suboptimal resultswithSuperScript IVVILO eventhought theyclaim to be‘compatible’
2 Copy Study 3 Master Mixes Applied Biosystems PowerTrack SYBR Green Master Mix BioRad SSoAdvanced Supermix Qiagen QuantiNova SYBR Green PCR Kit RNAseP Assay CEPH gDNA diluted down to 2 copies Run Protocol: Fast on QuantStudio 5 384-well block annealing Tm 60oC15
Product configurationsPowerTrack SYBR Global List ListGreen Master Mix PricePricePer/ml16A46012 (1ml) 91 91A46109 (5ml) 387 77A46110 (2 x 5ml) 709 73A46111 (5 x 5ml) 1687 70A46112 (10 x 5ml) 3358 67A46113 (50ml) 3260 65Compatible with: Invitrogen SuperScript IV VILO Master Mixreverse transcription
PowerTrack SYBR Green Master Mix: Key Technical SpecificationsGene expression analysis(Microbe detection,ChIP DNA analysis)ApplicationsRecommended master mixApplied Biosystems PowerTrack SYBR Green Master MixSensitivityBestSpecificityBetterPipetting tracking dyeHot startCycling modeCarryover contamination controlInstrument compatibilityRecommended primerconcentrationRecommended primer TmYesAntibody-mediatedStandard ( 60 min) orFast ( 30 min)Includes heat-labile UNG and a blend of dTTP/dUTPCompatible with all Applied Biosystems real-time PCR instruments,Bio-Rad CFX (96 and 384), and Roche LightCycler 480,300-800 nM nM55 – 65C
To learn more and request a free sample*Visit www.thermofisher.com/sybr Technical specifications See how PowerTrack SYBR Green Master Mix stacks up against other suppliers Request a free sample*No purchase necessary. Available to life sciences professionals 21 years and older in most countries. Offer will apply to sample requests until promotional samplesare depleted. Sample size: 1 mL. Limit one sample per laboratory. Offer void where prohibited by federal, state, provincial, or local laws or regulation oragency/institutional policy. Other restrictions may apply. View the privacy policy.18
Appendix Experimental conditions Additional performance data19
Experimental ConditionsSYBR Green Master MixesSamplesPowerTrack SYBR Green Master MixUHR cDNA concentration: 0.1 to 100,000pg/reaction by factors of 10PowerUp SYBR Master MixLuminaris Color HiGreen qPCR Master Mix, lowROXBio-Rad SsoAdvanced Universal SYBR GreenSupermixTakara TB Green Premix Ex Taq II (Tli RNase HPlus)ProMega GoTaq qPCR Master MixKAPA SYBR FAST qPCR Kits; Low Rox10 ml reactions run in quadruplicate4 cDNA Synthesis KitsSuperScript IV ViloRT KitBioRad iScriptHigh Capacity cDNA Qiagen QuantiTectSynthesis KitRT Kit4 Assays (400 nM final)PGK1, ARL1, GAPDH, SNF8, DF, C1orf19Qiagen QuantiNova SYBR Green PCR KitQiagen QuantiFast SYBR Green PCR KitInstrument/Run MethodBioLine (Meridian Bioscience) SensiFAST SYBR Lo-ROX KitQuantStudio 5 Real-time PCR Systemwith 384 well blockQuanta Biosciences PerfeCTa SYBR GreenFastMix Low ROX Reaction MixesThermal cycling performed asrecommended by each manufacturerAll assays had designed annealingtemperature of 60 degreesToyobo SYBR Green Realtime PCR Master Mix
SNF8ARL1PGK1Melt Curve Data shows PowerTrack SYBR Green Master Mix is specific21PowerTrackPerFecta PerfectaKapa KapaTakara GoTaq
PowerTrack SYBR Green Master Mix works efficiently with SuperScript IV VILO Master Mix reversetranscription kit and performs better compared to competitor mixes.GAPDH AssayQuantiNova .SsoAdv. PowerTrackDF AssayRT kit compatibility and Better Performance Better data
Thank youFor Research Use Only. Not for use in diagnostic procedures. 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientificand its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Sso AdvancedUniversal SYBR Green Supermix is a trademark of Bio-Rad Laboratories, TB Green Premix Ex Taq II is a trademark of Takara, GoTaq qPCR Master Mix is a trademark ofProMega, SYBR FAST qPCR Kits is a trademark of Kapa, QuantiNova SYBR Green PCR Kit and QuantiFast SYBR Green PCR Kitare trademarks of Qiagen, SensiFAST SYBR is a trademark of BioLine (Meridian Bioscience), PerfeCTa SYBR Green FastMix is a trademark of Quanta Bioscience andToyobo SYBR Green Realtime PCR Master Mix is a trademark of Toyoba.23
PowerTrack SYBR Green Master Mix For gene expression analysis customers who need broad dynamic range and to minimize pipetting mistakes without compromising sensitivity and specificity, PowerTrack SYBR Green Master Mix contains a tracking dye allowing users to know where they have pipetted: Challenge Benefit How 25% of poor real-time PCR data
