USB VeriQuest Fast SYBR Green qPCR Master Mix with ...€¦ · USB VeriQuest Fast SYBR Green qPCR...

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

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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