USB VeriQuest Fast SYBR Green qPCR Master Mix with ...€¦ · USB VeriQuest Fast SYBR Green qPCR...
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Applications: n Amplification from genomic DNA or cDNA input n Gene expression validation Features: n Uses fast mode thermal cycling conditions for results in one- third of the time compared to standard run protocols n One-tube master mix with Fluorescein Reference Dye included—just add template, primers and water n Reproducibility and consistency over a broad dynamic range: 8 orders of magnitude linear detection range (Fig. 1) n High specificity with exceptional performance on challenging GC-rich regions (Fig. 2) n Sensitivity and precision with limited targets (Fig. 3) n Stable at room temperature for 72 hours in a pre-assembled reaction (Fig. 4) n Contains dUTP and Uracil-DNA Glycosylase (UDG or UNG) for carry-over contamination prevention USB VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein is optimized for SYBR Green detection on all instru- ments that utilize Fluorescein as a passive reference dye. This master mix can be run using Fast mode cycling protocols, provid- ing results in one-third of the time when compared to standard real-time PCR reagents. The 2X ready-to-use master mix con- tains hot start VeriQuest Taq DNA Polymerase, MgCl 2 , ultrapure nucleotides with an optimized dUTP:dTTP ratio, Uracil-DNA Glycosylase (UDG or UNG), SYBR Green, and Fluorescein Passive Reference Dye in a proprietary reaction buffer. The hot start Taq polymerase has no polymerase activity prior to the initial heat activation step which allows reaction assembly at room tempera- ture. The mix offers higher specificity and sensitivity, virtually eliminating non-specific primer amplification which can nega- tively affect the efficiency and accuracy of the data (Fig. 2). Since the mix contains dUTP and UDG, carry-over contamination pre- vention can be performed prior to amplification. VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein offers the same sensitivity and specificity found with our standard mode mixes with results in a fraction of the time. VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein shows exceptional efficiency and high specificity on challenging templates such as high GC- and AT-rich regions (Fig. 2). The opti- mized formulation ensures no sacrifice in quality for increased speed. Performance comparisons highlight the sensitivity and reliable performance of this platform independent solution. The sensitivity of the master mix allows for discrimination from a 1.33-fold difference in gene target amount detected. In Fig. 2, a 1.33 to 10-fold dilution series of 10 ng to 1 ng of cDNA reverse- transcribed from HeLa total RNA were amplified with an effi- ciency of >99%. VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein is highly stable and easy to work with. It remains stable at room temperature for 72 hours in a pre-assembled reaction and can be stored at 4°C for convenient handling. The mix also allows for room temperature reaction set-up. The speed and stability make VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein ideal for high-throughput handling. USB ® VeriQuest ® Fast SYBR ® Green qPCR Master Mix with Fluorescein (2X) Fig. 1. Linear detection range Linear detection range of VeriQuest Fast SYBR Green qPCR Master Mix. Amplification plot and standard curve from real-time PCR for a dilution series of a synthetic target with starting amounts of 10 9 copies amplified in four replicate reactions using the ABI 7500 Real-Time PCR System and GAPDH primers. Reaction performed using fast mode cycling with 5 min- utes activation at 95°C. The amplification process was linear over eight orders of magnitude.
Transcript of 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
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
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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
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