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Applications:n AmplificationfromgenomicDNAorcDNAinputn Geneexpressionvalidation
Features:n Usesfastmodethermalcyclingconditionsforresultsinone-
thirdofthetimecomparedtostandardrunprotocolsn One-tubemastermixwithFluoresceinReferenceDye
included—justaddtemplate,primersandwatern Reproducibilityandconsistencyoverabroaddynamicrange:
8ordersofmagnitudelineardetectionrange(Fig.1)n Highspecificitywithexceptionalperformanceonchallenging
GC-richregions(Fig.2)n Sensitivityandprecisionwithlimitedtargets(Fig.3)n Stableatroomtemperaturefor72hoursinapre-assembled
reaction(Fig.4)n ContainsdUTPandUracil-DNAGlycosylase(UDGorUNG)for
carry-overcontaminationprevention
USBVeriQuestFastSYBRGreenqPCRMasterMixwithFluoresceinisoptimizedforSYBRGreendetectiononallinstru-mentsthatutilizeFluoresceinasapassivereferencedye.ThismastermixcanberunusingFastmodecyclingprotocols,provid-ingresultsinone-thirdofthetimewhencomparedtostandardreal-timePCRreagents.The2Xready-to-usemastermixcon-tainshotstartVeriQuestTaqDNAPolymerase,MgCl2,ultrapurenucleotideswithanoptimizeddUTP:dTTPratio,Uracil-DNAGlycosylase(UDGorUNG),SYBRGreen,andFluoresceinPassiveReferenceDyeinaproprietaryreactionbuffer.ThehotstartTaqpolymerasehasnopolymeraseactivitypriortotheinitialheatactivationstepwhichallowsreactionassemblyatroomtempera-ture.Themixoffershigherspecificityandsensitivity,virtuallyeliminatingnon-specificprimeramplificationwhichcannega-tivelyaffecttheefficiencyandaccuracyofthedata(Fig.2).SincethemixcontainsdUTPandUDG,carry-overcontaminationpre-ventioncanbeperformedpriortoamplification.VeriQuestFastSYBRGreenqPCRMasterMixwithFluoresceinoffersthesamesensitivityandspecificityfoundwithourstandardmodemixeswithresultsinafractionofthetime. VeriQuestFastSYBRGreenqPCRMasterMixwithFluoresceinshowsexceptionalefficiencyandhighspecificityonchallengingtemplatessuchashighGC-andAT-richregions(Fig.2).Theopti-mizedformulationensuresnosacrificeinqualityforincreasedspeed.Performancecomparisonshighlightthesensitivityandreliableperformanceofthisplatformindependentsolution.
Thesensitivityofthemastermixallowsfordiscriminationfroma1.33-folddifferenceingenetargetamountdetected.InFig.2,a1.33to10-folddilutionseriesof10ngto1ngofcDNAreverse-transcribedfromHeLatotalRNAwereamplifiedwithaneffi-ciencyof>99%. VeriQuestFastSYBRGreenqPCRMasterMixwithFluoresceinishighlystableandeasytoworkwith.Itremainsstableatroomtemperaturefor72hoursinapre-assembledreactionandcanbestoredat4°Cforconvenienthandling.Themixalsoallowsforroomtemperaturereactionset-up.ThespeedandstabilitymakeVeriQuestFastSYBRGreenqPCRMasterMixwithFluoresceinidealforhigh-throughputhandling.
USB®VeriQuest®FastSYBR®GreenqPCRMasterMixwithFluorescein(2X)
Fig. 1. Linear detection range
LineardetectionrangeofVeriQuestFastSYBRGreenqPCRMasterMix.Amplificationplotandstandardcurvefromreal-timePCRforadilutionseriesofasynthetictargetwithstartingamountsof109copiesamplifiedinfourreplicatereactionsusingtheABI7500Real-TimePCRSystemandGAPDHprimers.Reactionperformedusingfastmodecyclingwith5min-utesactivationat95°C.Theamplificationprocesswaslinearovereightordersofmagnitude.
VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein (2X)
Product code Pack size
75675 1mlfor100reactions(20µl)
5mlfor500reactions(20µl)
2x5mlfor1,000reactions(20µl)
5x5mlfor2,500reactions(20µl)
10x5mlfor5,000reactions(20µl)
Fig. 2. Specificity with amplification of GC rich targets
Meltcurveanalysisshowingampli-ficationresultsofCRAT,CarnitineO-Acetyltransferase,andAGT,Angiotensinogen.Resultsshowcomparisonsagainstcompetitormastermixes.
VeriQuest Fast SYBR
ABI Fast SYBR
Bio-Rad iTaq™ Fast SYBR
Bio-Rad SsoFast™ EvaGreen®
CRAT 60.5% GC
AGT 60.7% GC
Fig. 3. High sensitivity and precision in limited target quantification
Amplificationplot(left)andstandardcurve(right)fromreal-timePCRfora1.33to10-folddilutionseriesof10ngto1ngofcDNAsreverse-transcribedfromHeLatotalRNA.
GAPDH Kras
10 43 2 1 ng RNA
10 4 3 2 1 ng RNA
GAPDH2y = -3.3773x + 22.224
EPCR = 97.7%R² = 0.9955
Krasy = -3.2461x + 30.591
EPCR = 103.3%R² = 0.9993
151719212325272931
-0.2 0 0.2 0.4 0.6 0.8 1 1.2
Ct
log input
GAPDH2 Kras
Affymetrix, Inc.USB® Products26111MilesRoadCleveland,Ohio44128Tel:800-321-9322|216-765-5000Fax:800-535-0898|[email protected]
Affymetrix UK Ltd.USB® ProductsVoyager,MercuryPark,WycombeLane,WooburnGreen,HighWycombeHP100HH,UKTel:+44(0)1628552600Fax:+44(0)[email protected]
Affymetrix Pte Ltd.USB® Products7GulCircle,#2M-01KeppelLogisticsBuildingSingapore629563Tel:+6563957310Fax:[email protected]
72 hours y = -3.2678x + 21.44
EPCR = 102.3%R² = 0.9999
0 hoursy = -3.2774x + 21.268
EPCR = 101.9%R² = 1
14
19
24
29
34
39
-4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0
Ct
log ng input72 hours 0 hours
15
20
25
30
35
-4.0 -2.0 0.0 2.0
Ct
log ng input
B2M
ABI Fast SYBR
Bio-Rad iTaq Fast
VeriQuest Fast SYBR
VeriQuest Fast SYBRy = -3.1802 + 23.205EPCR = 106.3%R2 = 0.9999
Bio-Rad iTaq Fast SYBRy = -3.3764 + 22.908EPCR = 97.8%R2 = 0.9991
ABI Fast SYBRy = -3.1984 + 23.755EPCR = 105.4%R2 = 0.9995
Fig. 4. VeriQuest Fast SYBR Green qPCR Master Mix stability
Fig. 5. Exceptional results with performance comparison
GAPDHwasdetectedfrompre-assembledPCRreactionsincubatedatroomtemperaturefor72hours.Resultswerecomparedtothefreshlypre-paredmix(0hours).
Standardcurvesfromreal-timePCRfora10-folddilutionseriesof10ngto1pgcDNAreverse-transcribedfromHeLatotalRNAamplifiedinduplicatereactionswithVeriQuestFastSYBRGreenqPCRMasterMix,Bio-RadiTaqFastSYBRGreenSupermixwithROX™,andABIFastSYBRGreenMasterMix.
ForResearchUseOnly.Notforuseindiagnosticprocedures.Affymetrix,USB,andVeriQuestareregisteredtrademarksofAffymetrix,Inc.Pleaseseeaffymetrix.com/trademarksforacompletelistofAffymetrixtrademarks.Allothertrademarksarethepropertyoftheirrespectiveowners.TaqDNAPolymerase—Purchaseofthisproductconveystothepurchaserthelimited,non-transferablerightundercertainpatentstouseonlythepurchasedamountofproductforthepurchaser’sowninternalresearch.Norightunderanyotherpatentclaimsorforanyotheruseisconveyedexpressly,byimplication,orbyestoppel.FurtherinformationonpurchasinglicensesmaybeobtainedbycontactingOut-licensingatThermoFisherScientific,5791VanAllenWay,Carlsbad,California92008;outlicensing@lifetech.com.SYBRisaregisteredtrademarkofMolecularProbes,Inc.andisprovidedunderanagreementwithMolecularProbes,Inc.
©2016Affymetrix,Inc.Allrightsreserved. Rev06/15/16P/NUSB04974Rev.4