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Technical Data SheetKAPA SYBR FAST One-StepqRT-PCR Master Mix (2X) KitKR0393 – v5.16Product DescriptionThe KAPA SYBR FAST One-Step qRT-PCR Master Mix(2X) Kit is a sensitive and convenient solution for real-timePCR using RNA as template. All kits contain, at minimum,KAPA SYBR FAST Master Mix (2X), KAPA RT Mix (50X),and dUTP (10 mM). The KAPA RT Mix comprises wildtype M-MuLV Reverse Transcriptase and RNase Inhibitorand is optimized for rapid one-step, one-tube RNAquantification.KAPA SYBR FAST qPCR Master Mix (2X) contains a novelDNA polymerase engineered via a process of directedevolution. The result is a unique enzyme, specificallydesigned for high-performance real-time PCR using SYBRGreen I dye chemistry. The 2X master mix is a ready-to-usecocktail containing all components for the amplificationand detection of cDNA, except primers and template.The use of dUTP at the recommended final concentrationresults in amplicons that can be degraded using UracilDNA Glycosylase (UDG). UDG treatment is performed insubsequent reactions in order to minimize carryover PCRcontamination downstream. Use of dUTP in this system isoptional—UDG is not supplied in the kit.Ensure that the correct KAPA SYBR FAST qPCR MasterMix (2X) is used, in accordance with the reference dyerequirements (if any) of the qPCR instrument (Table 1).Product ApplicationsKAPA SYBR FAST One-Step qRT-PCR Master Mix (2X)Kits are ideally suited for: gene expression analysis;Kapa/Roche Kit Codes and ComponentsKAPA SYBR FAST One-StepqRT-PCR Master Mix (2X)UniversalKK4650 – 07959613001(100 x 20 µL reactions)qPCR Master Mix (2X)ROX High Reference Dye (50X)ROX Low Reference Dye (50X)dUTP (10 mM)KAPA RT Mix (50X)KK4651 – 07959621001(500 x 20 µL reactions)KAPA SYBR FAST One-StepqRT-PCR Master Mix (2X)ABI Prism KK4660 – 07959656001(100 x 20 µL reactions)qPCR Master Mix (2X) withROX High incorporateddUTP (10 mM)KAPA RT Mix (50X)KAPA SYBR FAST One-StepqRT-PCR Master Mix (2X)optimized for LightCycler 480qPCR Master Mix (2X) withno passive reference dyedUTP (10 mM)KAPA RT Mix (50X)KAPA SYBR FAST One-StepqRT-PCR Master Mix (2X)Bio-Rad iCyclerqPCR Master Mix (2X) withfluorescein incorporateddUTP (10 mM)KAPA RT Mix (50X)KK4652 – 07959630001(1000 x 20 µL reactions)KK4661 – 07959664001(500 x 20 µL reactions)KK4662 – 07959672001(1000 x 20 µL reactions)KK4680 – 07959753001(100 x 20 µL reactions)KK4681 – 07959761001(500 x 20 µL reactions)KK4682 – 07959770001(1000 x 20 µL reactions)KK4670 – 07959702001(100 x 20 µL reactions)KK4671 – 07959729001(500 x 20 µL reactions)KK4672 – 07959737001(1000 x 20 µL reactions)Quick Notes This kit contains wild-type M-MuLV and anengineered enzyme optimized for qPCR usingSYBR Green I dye chemistry. The 2X master mix contains a proprietary buffer.Together with the novel enzyme, this improvesamplification efficiency of both GC- and AT-rich targets. gene knockdown validation; Use only gene-specific primers for one-step qRT-PCR. microarray validation; Optimal cDNA synthesis is achieved at 42 C for 5 min. low copy gene detection; and 3 min at 95 C is sufficient for RT inactivation andDNA polymerase activation. RNAi and miRNA research. For 3-step cycling, use 20 sec for primer annealingand 1 sec for extension/data acquisition at 72 C. Do not exceed 25 μL reaction volumes.Effective date: October 2016For Research Use Only. Not for use in diagnostic procedures.

KAPA SYBR FAST One-Step qRT-PCRMaster Mix (2X) KitProduct SpecificationsShipping and StorageKAPA SYBR FAST qRT-PCR Master Mix (2X) Kits areshipped on dry ice or ice packs, depending on thedestination country. Upon receipt, immediately store allcomponents at -20 C in a constant-temperature freezer.When stored under these conditions and handled correctly,the kit components will retain full activity until the expirydate indicated on the kit label.HandlingAlways ensure that components have been fully thawedand thoroughly mixed before use. The KAPA SYBRFAST qPCR Master Mix (2X) may not freeze solidly, evenwhen stored at -20 C. The KAPA RT Mix is temperaturesensitive, and should be stored at -20 C and kept on iceduring use.The SYBR Green I dye contained in the KAPA SYBRFAST qPCR Master Mix (2X) and ROX/fluorescein dyes(depending on kit configuration) are light sensitive.Exposure to direct light for an extended period of time willresult in loss of fluorescent signal intensity.Technical Data SheetTable 1. Instrument Compatibility*InstrumentReference DyeApplied Biosystems 5700, 7000,7300, 7700, 7900HT, StepOne , andStepOnePlus ROX High500 nMApplied Biosystems 7500, ViiA 7,QuantStudio instruments, AgilentMx3000P , Mx3005P and Mx4000 ROX Low50 nMRotor-Gene instruments, DNA EngineOpticon , Opticon 2, Chromo 4 Real-Time Detector, Mastercycler ep realplex, Smart Cycler , RocheLightCycler 480, 96, Nano, 1.5/2.0**,Bio-Rad CFX96, Illumina Eco No ROXBio-Rad iCyclersFluorescein* For instruments not listed here, please contact Technical Support atkapabiosystems.com/support for more information.** The Roche LightCycler 1.5/2.0 capillary instruments require the additionof unacetylated BSA to the qPCR reaction at a final concentration of250 ng/µL in order to prevent the DNA polymerase and template from binding tothe glass capillaries.KAPA SYBR FAST qPCR Master Mix (2X) is stablethrough 30 freeze-thaw cycles. Ensure that all reagentsare stored protected from light at -20 C when not in use.When protected from light, reagents are stable in the darkat 4 C for at least one week and may be stored at thistemperature for short-term use provided that they do notbecome contaminated with microbes and/or nucleases.Quality ControlAll kit components are subjected to stringent functionalquality control, are free of detectable contaminating exoand endonuclease activity, and meet strict requirementswith respect to DNA contamination. Please contactTechnical Support at kapabiosystems.com/support formore information.2For Research Use Only. Not for use in diagnostic procedures.

KAPA SYBR FAST One-Step qRT-PCRMaster Mix (2X) KitImportant ParametersTemplate RNAStarting template of purified total RNA can range between1 pg – 100 ng per 20 μL reaction. Using greater amountsof template may increase the background fluorescence,which reduces linearity of standard curves afterbackground subtraction. Digest purified RNA with RNasefree DNase I to remove contaminating genomic DNAwhich can act as template during PCR. DNase treatmentshould be performed according to manufacturer’sinstructions. Treated RNA should be stored at -20 C or-80 C in RNase-free water. Multiple freeze-thaws of RNAshould be avoided.PrimersCareful primer design and purification (HPLC-purifiedprimers are recommended) will minimize loss insensitivity due to non-specific amplification. This effectbecomes more prominent at low target concentrations.To maximize the sensitivity of the assay, use the lowestprimer concentration that does not compromise reactionefficiency (50 – 400 nM of each primer). For optimal results,design primers that amplify PCR products 70 – 200 bp inlength. Use appropriate primer design software to designprimers with a melting temperature (Tm) of approximately60 C to take advantage of two-step cycling. Primers mustbe carefully designed to avoid detection and amplificationof genomic DNA, which would lead to inaccurate mRNAquantification. To prevent gDNA amplification, designforward and reverse primers from different exons, or tospan exon-intron boundaries.KAPA SYBR FAST DNA PolymeraseKAPA SYBR FAST DNA Polymerase is an engineeredversion of Taq DNA polymerase, designed specifically forreal-time PCR using SYBR Green I chemistry. KAPA SYBRFAST DNA Polymerase displays no enzymatic activityat ambient temperature. This prevents the formation ofmisprimed products and primer-dimers during reactionsetup prior to the first denaturation step and resultsin high PCR specificity and accurate quantification.The enzyme is activated during the initial denaturation stepof the PCR. The activation of the enzyme is complete after20 sec; however, complex targets may require up to 3 minfor optimal denaturation. The hot start feature obviates theneed to cool reactions during setup.KAPA RT MixKAPA RT Mix (50X) contains an optimized blend of wildtype M-MuLV Reverse Transcriptase and RNase Inhibitor.M-MuLV RT has a high affinity for RNA and is optimizedfor cDNA synthesis at 42 C. The RNase Inhibitorsafeguards against degradation of RNA target due toRNase contamination. KAPA RT Mix (50X) must be storedat -20 C as the enzymes are not thermostable, and mustbe worked with on ice at all times.For Research Use Only. Not for use in diagnostic procedures.Technical Data SheetdUTPUse of dUTP allows treatment with uracil-DNA glycosylase(UDG) if required to prevent carryover contaminationin subsequent reactions. dUTP can be used at finalconcentrations that range from 0.2 – 0.4 mM. Do not useUDG in one-step qRT-PCR, as the UDG will degrade thecDNA upon synthesis.ROX Reference DyeFor certain real-time cyclers, the presence of ROXreference dye compensates for non-PCR-relatedvariations in fluorescence detection. The fluorescencelevel of ROX reference dye does not change significantlyduring the course of real-time PCR, but provides astable baseline against which PCR-related fluorescentsignals are normalized. Thus, ROX dye compensates fordifferences in fluorescence detection between wells dueto slight variations in reaction volume or differences in wellposition. The presence of ROX dye in the master mix doesnot interfere with real-time PCR on any instrument, sincethe dye is not involved in the reaction and has an emissionspectrum different from that of SYBR Green I.Fluorescein Reference DyeThe use of fluorescein reference dye is necessary forBio-Rad iCycler iQ , iQ 5, and MyiQ instruments andis included in the KAPA SYBR FAST One-Step qRT-PCRMaster Mix (2X) for Bio-Rad iCycler at a final concentrationof 10 nM. The presence of the fluorescein dye in thismaster mix does not interfere with real-time PCR on anyinstrument, since the dye is not involved in the reaction.SYBR Green IKAPA SYBR FAST qPCR Master Mix (2X) contains anelevated, optimized concentration of the fluorescent dye,SYBR Green I. High signal intensities are achieved as aresult of increased tolerance to high concentrations ofSYBR Green I by the engineered KAPA SYBR FAST DNAPolymerase. SYBR Green I binds all double-stranded DNAmolecules, emitting a fluorescent signal on binding.Magnesium ChlorideKAPA SYBR FAST qPCR Master Mix (2X) contains anoptimized MgCl2 concentration. It is highly unlikelythat additional MgCl2 will improve reaction efficiencyor specificity.Melting Curve AnalysisFollowing real-time qPCR, melting curve analysis shouldalways be performed to identify the presence of primerdimers and analyze the specificity of the reaction. Programyour thermocycler according to the instructions provided.3