SMART PCR cDNA Synthesis Kit User Manual - genex.cl · PDF fileProtocol No. PT3041-1 Clontech Laboratories, Inc. Version No. PR6Z2173 3 SMART ™ PCR cDNA Synthesis Kit User Manual

SMART™ PCR cDNA Synthesis KitUser Manual

Cat. No. 634902 PT3041-1 (PR6Z2173)Published 22 December 2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 � Version No. PR6Z2173

SMART ™ PCR cDNA Synthesis Kit User Manual

I. Introduction 4

II. List of Components 8

III. Additional Materials Required 9

IV. General Considerations 10

V. SMART™ cDNA Synthesis for Library Construction 12

A.First-StrandcDNASynthesis 13

B. cDNAAmplificationbyLDPCR 14

C.dscDNAPolishing 15

VI. Analysis of Results for Library Construction 16

VII. SMART cDNA Synthesis Protocol 18 A.First-StrandcDNASynthesis 19

B. cDNAAmplificationbyLDPCR 20

VIII. Protocol for Clontech PCR-Select™ cDNA Subtraction 23 A.ColumnChromatography 23

B. RsaIDigestion 24

C.PurificationofDigestedcDNA 25

D. ControlsforClontechPCR-SelectcDNASubtraction 27

IX. Analysis for Clontech™ PCR-Select Subtraction 28

A.DeterminingtheOptimalNumberofPCRCycles 28

B. ColumnChromatography 29

C.RsaIDigestion 29

D. PurificationofDigestedcDNA 30

X. Troubleshooting Guide 31

A.First-StrandcDNASynthesisandSMARTPCR 31 Amplification

B. SpecialConsiderationsforLibraryConstruction 32

C.PreparationforClontechPCR-SelectcDNASubtraction 33

XI. References 34

XII. Related Products 35

Appendix: Virtual Northern Blots 36

Table of Contents

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List of Figures

Figure 1. FlowchartofSMARTtechnology 5

Figure 2. GuidetoSMARTcDNAsynthesisprotocols 6

Figure 3. ProtocolguideforSMARTcDNAsynthesisforlibraryconstruction 12

Figure 4. AnalysisofdscDNAsynthesizedforlibraryconstruction 17

Figure 5. ProtocolguideforSMARTcDNAsynthesisforPCR-SelectcDNAsubtractionandotherapplications 18

Figure 6. OptimizingPCRparametersforSMARTcDNAsynthesis 20

Figure 7. AnalysisforoptimizingPCRparameters 28

Figure 8. VirtualNorthernblotanalysisofcDNAfragmentsexpressedincellsproducingγ-globin 36

List of Tables

Table I. PCRcyclingparameters(libraryconstruction) 14

Table II. GuidelinesforsettingupPCR 21

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I. Introduction

TheSMART™PCRcDNASynthesisKitprovidesanovel,PCR-basedmethodforproducinghigh-qualitycDNAfromnanogramsoftotalorpolyA+RNA.SMARTtechnologyisespeciallyusefulforresearcherswhohavelimitedstartingmaterial,suchastotalRNAfromasmallsample.

SMART™ cDNA synthesis technologyAll commonly used cDNA synthesis methods rely on the ability of reversetranscriptase (RT) to transcribemRNA intosingle-stranded (ss) cDNA in thefirst-strandreaction.However,becauseRTcannotalwaystranscribetheentiremRNAsequence,the5'endsofgenestendtobeunder-representedincDNApopulations.ThisisoftenthecaseforlongmRNAs,especiallyifthefirst-strandsynthesisisprimedonlywitholigo(dT)primers,orifthemRNAhasapersistentsecondary structure. In the absence of RNA degradation, truncated cDNAmoleculespresentinlibrariesareoftenduetoRTpausingbeforetranscriptioniscomplete.Regardless,theSMARTmethodisabletopreferentiallyenrichforfull-lengthcDNAs.SMART cDNA synthesis starts with either total or poly A+ RNA. A modifiedoligo(dT) primer (the 3' SMART CDS Primer II A) primes the first-strandsynthesis reaction (Figure 1). When RT reaches the 5' end of the mRNA,the enzyme’s terminal transferase activity adds a few additional nucleotides,primarily deoxycytidine, to the 3' end of the cDNA. The SMART™ II AOligonucleotide,whichhasanoligo(G)sequenceatits3'end,base-pairswiththe deoxycytidine stretch, creating an extended template. RT then switchestemplatesandcontinuesreplicatingtotheendoftheoligonucleotide(Chenchiket al., 1998).Theresultingfull-length,single-stranded(ss)cDNAcontainsthecomplete5'endofthemRNA,aswellassequencesthatarecomplementaryto theSMARTOligonucleotide. IncaseswhereRTpausesbefore theendofthe template, theadditionof deoxycytidinenucleotides ismuch lessefficientthanwithfull-lengthcDNA-RNAhybrids,thuspreventingbase-pairingwiththeSMARTOligonucleotide.TheSMARTanchorsequenceandthepolyAsequenceserveasuniversalprimingsitesforend-to-endcDNAamplification.Therefore,cDNAwithoutthesesequencesduetoprematurelyterminatedcDNAscausedby incompleteRTactivity,contaminatinggenomicDNA,orcDNA transcribedfrompolyA–RNA,willnotbeexponentiallyamplified.However,truncatedRNAsthatarepresentinpoorqualityRNAstartingmaterialwill beamplified,whichwillcontaminatethefinalcDNAlibrary.

Synthesize SMART™ cDNA for a wide variety of applicationsThefirstkittofeatureSMARTtechnologyistheSMARTcDNALibraryConstructionKit(Cat.No.634901).Thiskitincludesthecomponentsfordirectionalcloningoffull-lengthcDNA.Toexpandtherangeofapplications,theSMARTPCRcDNASynthesisKit(Cat.No.634902;Figure2)wasintroducedshortlyafter.Thiskitallowsyoutosynthesizehigh-qualitycDNAforlibraryconstructionusingyourownvectorandligationreagents.OtherapplicationsincludeClontechPCR-Select™

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I. Introduction continued

Figure 1. Flow chart of SMART™ technology. The SMART II A Oligonucleotide,3'SMARTCDSPrimerIIA,and5'PCRPrimerIIAallcontainastretchofidenticalsequence(seeSectionIIforcompletesequenceinformation).

cDNASubtraction(Cat.No.637401),“Virtual”Northernblots,andprobegeneration.PleasenotethattheSMARTII™AOligonucleotideisspeciallyengineeredforuse with the PCR-Select method. cDNA generated using the SMART cDNALibraryConstructionKitcannotbeusedforPCR-SelectcDNAsubtraction.IntheSMARTlibraryconstructionprotocol,eachPCR-amplifiedcDNAmoleculehasanextraSMARTsequenceoneachendwhichdecreasestheefficiencyofsubtractionofamplifiedcDNA.

First-strand synthesis by RT

Amplify cDNA by LD PCRwith PCR primer

Poly A+ RNA

polyA 3'

SMART II Aoligonucleotide

CDS primer

Double-stranded cDNA

Template switching and extension by RT

polyA

polyAGGG

5'

GGG5'

GGG5'

CCC

dC tailing by RT

polyA

CCCGGG5'

Singlestep

5'

5'

5'




TheSMARTIIAOligonucleotideand3'SMARTCDSPrimerIIAprovidedintheSMARTPCRcDNASynthesisKiteachhaveanRsaIsitetofacilitateremovaloftheseidenticalsequencesfromthePCR-amplifiedcDNAmolecules.ThisUserManualincludestwoprotocolsforcDNAsynthesis.Theseprotocolshavebeendesignedtostrikeabalancebetweenmaintaininggenerepresentationandreducingnonspecificbackgroundamplification.Inthefirstprotocol(SectionsVandVI),undilutedfirst-strandsscDNA is subjected to the fewestpossiblenumberofPCRcycles.ThisprotocolisidealforcDNAlibraryconstruction,wherehighrepresentationismostimportant(Zhuet al.,2001).Inthesecondprotocol(SectionsVIIandVIII),thefirst-strandsscDNAtemplateisdilutedandmorePCRcyclesareperformed.Thisgreatlyreducesnonspecificamplification,whichiscrucialforPCR-SelectcDNAsubtractionandothernon-libraryapplications.Besuretochoosetheappropriateprotocolforyourapplication.TheSMARTcDNAsynthesismethodisnowoptimizedforrapidamplificationofcDNAends(RACE;Matzet al., 1999).TheSMART™RACEcDNAAmplificationKit(Cat.No.634914)integratesourMarathon®cDNAAmplificationKit(Chenchiket al., 1995;1996)withourSMARTcDNAsynthesistechnologyandallowsyoutoperformboth5'and3'RACEusingeitherpolyA+ortotalRNA.ClontechhasrigorouslytestedournewSMARTRACEKittoverifythatitperformsevenbetterthantheMarathonKit(January1999Clontechniques).

Figure 2. Guide to SMART™ cDNA synthesis protocols.Besuretofollowtheappropriateprotocolforyourapplication.

Total or poly A+ RNA

SMART ds cDNA SMART ds cDNA

SMART cDNA synthesis(Sections VII & VIII)

SMART cDNA synthesis(Sections V & VI)

Other applications

PCR-SelectcDNA

subtractionVirtual

Northern blotscDNA libraryconstruction




SMART™ cDNA synthesis for cDNA subtractionTheClontechPCR-SelectcDNASubtractionKit(Cat.No.637401)providesapowerfulmethodforidentifyingdifferentiallyexpressedgenes(Diatchenkoet al.,1996;Gurskayaet al.,1996).WhentotalRNAisusedforcDNAsynthesisbycon-ventionalmethods,ribosomalRNAistranscribedalongwiththepolyA+fraction,evenifsynthesisisoligo(dT)-primed.IfthiscDNAisusedwiththePCR-SelectKit,theexcessofribosomalRNAandlowconcentrationofcDNAcorrespondingto thepolyA+ fractionresults in inefficientsubtractivehybridization.However,cDNAgeneratedusingtheSMARTPCRcDNASynthesisKitcanbedirectlyusedforPCR-Selectsubtraction—eveniftotalRNAwasusedasstartingmaterial.

Virtual Northern blots and probesTheSMARTPCRcDNASynthesisKitmayalsobeusefulforresearcherswhowishtoanalyzetranscriptsizeandexpressionpatternsbyhybridizationbutlacksufficientpolyA+ortotalRNAforNorthernblots.ThisisespeciallyimportantforresearcherswhohaveisolatedclonesusingtheClontechPCR-Select™KitandwhoalsoneedtoconfirmthedifferentialexpressionofcorrespondingmRNAs.“Virtual”NorthernblotscanbegeneratedusingSMARTcDNAinsteadoftotalorpolyA+RNA(Endegeet al.,1999),andcangiveinformationsimilartothatprovidedbystandardNorthernblots.FormoreinformationonVirtualNorthernblots,pleaseseetheAppendix.OtherapplicationsforSMARTcDNAincludepreparingprobesforhybridizationtohigh-densitycDNAorgenomicDNAarrays(Pietuet al.,1996)orforthecDNAselection-basedpositional cloningmethod (Morganet al., 1992).Please seethesereferencesformoreinformationabouttheseapplications.

Advantage® 2 PCR Kit and PowerScript™ Reverse TranscriptaseWestronglyrecommendtheuseoftheAdvantage2PCRKits(Cat.Nos.639206&639207)forPCRamplification.ThesekitsincludetheAdvantage2PolymeraseMix,whichhasbeenspeciallyformulatedforefficient,accurate,andconvenientamplificationofcDNAtemplatesbylong-distancePCR(LDPCR;Barnes,1994).The Polymerase Mix is comprised ofTITANIUM™ Taq DNA Polymerase—anuclease-deficientN-terminaldeletionofTaqDNApolymeraseplusTaqStart™Antibodytoprovideautomatichot-startPCR(Kellogget al.,1994)—andaminoramountofaproofreadingpolymerase.Thiscombinationallowsyoutoefficientlyamplifyfull-lengthcDNAswithasignificantlylowererrorratethanthatofcon-ventionalPCR(Barnes,1994).Each SMART kit also includes PowerScript™ Reverse Transcriptase, apoint mutant of Moloney murine leukemia virus (MMLV) reverse transcrip-tase (RT). PowerScript RT lacks RNase H activity, but retains wild-typepolymerase activity, so it can synthesize longer cDNA fragments than wild-type MMLV RT. Our rigorous purification method also ensures that eachPowerScriptpreparationisnotcontaminatedwithRNaseandDNase.



II. List of Components

StoreCHROMASPINandMicrofiltrationcolumnsatroomtemperature.StoreRNAandSMARTIIAOligoat–70°C.Storeallotherreagentsat–20°C.

ForimportantinformationabouttheuseofSMARTtechnology,pleasereadtheNoticetoPurchaserattheendofthisUserManual.Box 1:• 7 µl SMART II™ A Oligonucleotide(12µM) 5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3' RsaI

• 7 µl 3' SMART™ CDS Primer II A(12µM) 5'-AAGCAGTGGTATCAACGCAGAGTACT(30)VN-3' (N=A,C,G,orT;V=A,G,orC)RsaI

• 7 µl PowerScript™ Reverse Transcriptase • 200 µl 5X First-Strand Buffer 250mM Tris-HCl(pH8.3) 375mM KCl 30mM MgCl2• 100 µl 5' PCR Primer II A(12µM) 5'-AAGCAGTGGTATCAACGCAGAGT-3'• 70 µl dNTP Mix(10mMofeachdNTP)• 200 µl Dithiothreitol (DTT;20mM)• 5 µl Control Human Placental Total RNA (1µg/µl)• 1 ml Deionized H2OBox 2:• 7 CHROMA SPIN™ 1000 Columns• 7 Microfiltration Columns (0.45μm)



III. Additional Materials Required

Thefollowingreagentsarerequiredbutnotsupplied:

First-strand cDNA synthesis and SMART™PCR cDNA amplification• Advantage® 2 PCR Kit (Cat.Nos.639206&639207)• [Optional]Mineral oil (SigmaCat.No.M3516) • Phenol:chloroform:isoamyl alcohol(25:24:1) Prepareasfollows: 1.Meltphenol. 2.Equilibratewithanequalvolumeofsterilebuffer(50mMTris[pH7.5],

150mMNaCl,1mMEDTA). 3. Incubatethemixtureatroomtemperaturefor2–3hr. 4.Removeanddiscardthetoplayer. 5.Addanequalvolumeofchloroform:isoamylalcoholtotheremaining

layer.Mixthoroughly.Removeanddiscardthetoplayer. 6.Storethebottomlayerofphenol:chloroform:isoamylalcohol(24:1)at

4°Cawayfromlightforamaximumoftwoweeks.• TE buffer(10mMTris[pH7.6],1mMEDTA)• Ethanol• 4 M Ammonium acetate (pH7.0)• DNA size markers (1-kbDNAladder)• 50X TAE electrophoresis buffer 242.0g Trisbase 57.1ml glacialaceticacid 37.2g Na2EDTA•2H2O AddH2Oto1L.

ds cDNA polishing for library construction• Proteinase K(20µg/µl;RocheAppliedScienceCat.No.0161519)• T4 DNA Polymerase (NewEnglandBiolabsCat.No.M0203S)

Purification for Clontech PCR-Select™ cDNA Subtraction• 1X TNE buffer (10mMTris-HCl[pH8],10mMNaCl,0.1mMEDTA)• NucleoTrap® Purification Kit(Cat.No.636020) NucleoTrapSuspension 80mlBufferNT2 16mlBufferNT3



PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.

• Thiskitisdesignedfortheconstructionofhigh-qualitySMARTcDNAforavarietyofapplications.ThisUserManualprovidestwoprotocolsforcDNAsynthesis:oneforcDNAlibraryconstruction(SectionsVandVI),andoneforotherapplications, includingClontechPCR-SelectcDNASubtraction(SectionsVIIandVIII). Be sure to follow the appropriate protocol for your application (see Figure 2).

• Propertemplateswitching,whichisessentialtotheSMARTtechnology,requirestheuseofanMMLVRNaseH–pointmutant(not deletionmutant)reversetranscriptasesuchasPowerScriptReverseTranscriptase(includedwitheachSMARTKit).

• Theprotocolshavebeenoptimizedforboth totalandpolyA+RNA.TheminimumamountofstartingmaterialforcDNAsynthesisis50ngoftotalRNAor25ngofpolyA+RNA.However,ifyourRNAsampleisnotlimiting,werecommendthatyoustartfrom1µgoftotalRNAor0.5µgofpolyA+RNAforcDNAsynthesis.

• Whateveryourapplicationmaybe,thesuccessofyourexperimentdependsonthequalityofyourstartingsampleoftotalorpolyA+RNA.Thereareseveralproceduresavailable forRNA isolation (Chomczynski&Sacchi,1987;Farrell,1993;Sambrooket al.,2001).Inaddition,Clontechoffersseveral kits for the isolation of total RNA and subsequent isolation ofpolyA+RNA.Alternatively,youmaywishtouseoneofourPremiumPolyA+RNAs.Formoreinformation,visitourwebsiteatwww.clontech.com.

• Beforeyoubeginfirst-strandsynthesis,westronglyrecommendthatyouchecktheintegrityofyourRNAbyelectrophoresingasampleonaformaldehyde/agarose/EtBrgel.FormammaliantotalRNA,youshouldobservetwobrightbandsatapproximately4.5and1.9kb;thesebandsrepresent28Sand18SribosomalRNA,respectively.Theratioofintensitiesofthesebandsshouldbe1.5–2.5:1.IntactmammalianpolyA+RNAshouldappearasasmear(usually0.5–12kb)withfaint28Sand18SrRNAbands.Thesizedistributionmaybeconsiderablysmaller(0.5–3kb)fornonmammalianspecies(e.g.,plants,insects,yeast,andamphibians).Formoreinformation,seeSambrooket al.(2001).

• Wear gloves throughout the procedure to protect your RNA and cDNAsamplesfromdegradationbynucleases.

IV. General Considerations



IV. General Considerations continued

• Thefirsttimeyouusethiskit,youshouldperformcDNAsynthesiswiththeControlHumanPlacentalTotalRNAprovidedinthekit,inparallelwithyourexperimentalsample.Performingthiscontrolsynthesisatleastoncewillverifythatallcomponents(especiallythereversetranscriptase)areworkingproperlyandwillalsohelpyoutroubleshootanyproblemsthatmayarise.

• The cycling parameters in this protocol have been optimized using anauthorizedhot-lidthermalcycler.Optimalparametersmayvarywithdifferentthermalcyclersandtemplates.

• Toresuspendpelletsandmixreactions,gentlypipetthemupanddownandcentrifugethetubebrieflytodepositcontentsatthebottom.

• Vortexphenol:chloroformextractionstomix.

• Add enzymes to reaction mixtures last, and thoroughly incorporate theenzymebygentlypipettingthereactionmixtureupanddown.

• Donot increase theamountofenzymeaddedorconcentrationofDNAin the reactions. The amounts and concentrations have been carefullyoptimized.

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V. SMART™ cDNA Synthesis for Library Construction

Important: ThisprotocolisdesignedforsynthesizingSMARTcDNA for library construction.Forotherapplications,includingClontechPCR-SelectcDNAsub-traction,consulttheprotocolinSectionsVIIandVIII.

Figure 3. Protocol guide for SMART™ cDNA synthesis for library construction. Ifagarose/EtBrgelanalysisofthedscDNAindicatesthatmorecyclesareneeded,simplyreturnthereactiontothethermalcyclerforafewmorecycles,asdescribedintheTroubleshootingGuide(SectionX.B).


SMART™ ds cDNA (Section V.B)

cDNA library construction*

First-strand ss cDNA(Section V.A)

ds cDNA polishing*(Section V.C)

Agarose/EtBr gel analysis(Figure 4, Section VI)

Troubleshooting(Section X.A & B)

*Reagents for these procedures are not included in the SMART PCR cDNA Synthesis Kit.



V. SMART cDNA Synthesis for Library... continued

A. First-Strand cDNA Synthesis 1.Foreachsampleandcontrol,combinethefollowingreagentsinasterile

0.5-mlreactiontube:

1–3 µl RNAsample* (0.025–0.5µgofpolyA+or0.05–1µgoftotalRNA)

1µl 3'SMARTCDSPrimerIIA(12µM)1µl SMARTIIAOligonucleotide(12µM) xµl DeionizedH2O

5µl Totalvolume*Forthecontrolsynthesis,add1µl(1µg/µl)ofControlHumanPlacentalTotalRNA.

2.Mixcontentsandspinthetubebrieflyinamicrocentrifuge. 3. Incubatethetubeat72°Cfor2min. 4.Coolthetubeonicefor2min. 5.Centrifugethetubebrieflyinamicrocentrifugetocollectcontentsat

thebottom.

6.Addthefollowingtoeachreactiontube:2µl 5XFirst-StrandBuffer1µl DTT(20mM)1µl dNTPMix(10mMofeachdNTP)1µl PowerScriptReverseTranscriptase

7.Mixbygentlypipettingandspinthetubesbrieflyinamicrocentrifuge. 8. Incubatethetubesat42°Cfor1hrinanairincubator.

Note:Ifyouuseawaterbathorthermalcyclerforthisincubation,coverthereactionmixturewithonedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

9.Placethetubeonicetoterminatefirst-strandsynthesis. 10. IfyouplantoproceeddirectlytothePCRstep(SectionV.B),transfer

a 2-µl aliquot from the first-strand synthesis to a clean, prechilled,0.5-mlreactiontube.Placetubeonice.Ifyouusedmineraloilinyourfirst-strandreactiontube,becarefultotakethealiquotfromthebottom ofthetubetoavoidtheoil.

11.Anyfirst-strandreactionmixturethatisnotusedrightawayshouldbeplacedat–20°C.First-strandcDNAcanbestoredat–20°Cforuptothreemonths.




B. cDNA Amplification by LD PCR Table Iprovidesguidelines for theoptimalnumberof thermalcycles for

agivenamountoftotalorpolyA+RNAusedinthefirst-strandsynthesis.These guidelines were developed using the Control Human PlacentalTotalRNAandanauthorizedhot-lid thermalcycler;optimalparametersmay vary with different templates and thermal cyclers. Use the fewest cycles possible; overcycling may yield nonspecific PCR products. Ifnecessary,undercyclingcanbeeasilyrectifiedbyplacingthereactionbackin thethermalcycler fora fewmorecycles(seeTroubleshootingGuide,SectionX.B).

tablei:pcrcyclingparameters(libraryconstruction)

Total RNA Poly A+ RNA (µg) (µg) Number of Cycles

1.0–2.0 0.5–1.0 13–15

0.5–1.0 0.25–0.5 15–18

0.25–0.5 0.125–0.25 18–21

0.05–0.25 0.025–0.125 21–24

1.Preheatathermalcyclerto95°C.

2.PrepareaMasterMixforallreactiontubes,plusoneadditionaltube.Combinethefollowingcomponentsintheordershown:perrxn

80µl DeionizedH2O 10µl 10XAdvantage2PCRBuffer 2µl 50XdNTPMix(10mMofeachdNTP) 4µl 5'PCRPrimerIIA(12µM) 2µl 50XAdvantage2PolymeraseMix 98µl Totalvolume 3.Mix well by vortexing and centrifuge the tube briefly in a

microcentrifuge. 4.Aliquot 98 µl of the Master Mix into each reaction tube from Step

A.10. 5.Mixcontentsbygentlyflickingthetubes.Centrifugetubesbrieflyina

microcentrifuge. 6.Capthetube,andplaceitinthepreheatedthermalcycler.Ifnecessary,

overlaythereactionmixturewith2dropsofmineraloil.

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7.Commencethermalcyclingusingthefollowingprogram: •95°C 1min •xcycles*: 95°C 15sec 65°C 30sec 68°C 6min

*ConsultTableIforguidelines.

8.Whenthecyclingiscompleted,electrophorese5µlofeachsampleona1.1%agarose/EtBrgelin1XTAEbuffer.Forcomparison,Figure4showsthecharacteristicgelprofileofdscDNAsynthesizedfromtheControlHumanPlacentalTotalRNA(SectionVI).

C. ds cDNA Polishing We recommend the following procedure for polishing the ends of

SMARTcDNAsforconstructinglibraries.

1.Combine 50 µl (2–5 µg) of the amplified ds cDNA with 2 µl ofProteinaseK(20µg/µl)inasterile0.5-mlmicrocentrifugetube.StoretheremainderofthePCRmixtureat–20°C.Note: ProteinaseKtreatment isnecessarytoinactivatetheDNApolymeraseactivitybeforeproceedingwiththeligationsteps.

2.Mixcontentsandspinthetubebriefly. 3. Incubateat45°Cfor1hr.Spinthetubebriefly. 4.Heatthetubeat90°Cfor8–10mintoinactivatetheProteinaseK. 5.Chillthetubeinicewaterfor2min. 6.Add3µl(15units)ofT4DNAPolymerase. 7. Incubatethetubeat16°Cfor30min. 8.Heatthetubeat72°Cfor10min. 9.Add27.5µlof4Mammoniumacetate. 10.Add~210µlofroomtemperature95%ethanol. 11.Mixthoroughlybyinvertingthetube. 12.Spin the tube immediately at 14,000 rpm for 20 min at room

temperature.Note: Donotchillthetubeat–20°Coronicebeforecentrifuging.Chillingthesamplewillresultincoprecipitationofimpurities.

13.Carefullyremovethesupernatant. 14.Washpelletwith80%ethanol. 15.Airdrythepellet(~10min)toevaporateresidualethanol. 16.AdddeionizedH2O to resuspend thepellet.Theamountaddedwill

dependonyourcDNAlibraryconstructionprotocol.Note:Thispreparationofblunt-endedcDNAmaynowbe ligated toanyadaptoryouchoose.ConsultyourprotocolforcDNAlibraryconstruction.



VI. Analysis of Results for Library Construction

Figure4showsatypicalgelprofileofdscDNAsynthesizedusingtheControlHumanPlacentalTotalRNAandtheSMARTprotocoloutlinedinSectionV.Thesampleshownwas takenafterStepV.B.8andrepresents“raw”cDNAbeforepolishing.Typicalresults,indicativeofasuccessfulPCR,shouldhavethefollowingcharacteristics: 1. AmoderatelystrongsmearofcDNAfrom0.5to6kb ComparetheintensityofthebandingpatternofyourPCRproducttothe

1-kbDNAladdersizemarker(0.1µgrunonthesamegel).ForcDNAmadefromallmammalianRNAsources,theoverallsignalintensity(relativetothemarkerDNA)shouldberoughlysimilartothatshownforthecontrolexperimentinFigure4.IftheintensityofthecDNAsmearismuchstrongerthanthatshownforthecontrol(relativeto0.1µgofsizemarker),especiallyif no bright bands are distinguishable, this may indicate that too manythermalcycleswereused—thatis,youhaveovercycledyourPCR(seeTroubleshootingGuide,SectionX.B).Ifthesmearismuchfainter(relativeto0.1µgofsizemarker)andthesizedistributiongenerallylessthan4kb,thentoofewthermalcycles(i.e.,PCRundercycling)maybetheproblem(seeTroubleshootingGuide,SectionX.B).

2. Severalbrightbandscorrespondingtoabundanttranscripts ThepatternofbrightbandsshowninFigure4ischaracteristicoftheds

cDNAsynthesizedfromtheControlHumanPlacentalTotalRNAusingtheprotocoloutlinedinSectionV.AsindicatedbythearrowinFigure5,youshouldobserveastrong,distinctbandat900bp.AverystrongsmearofcDNAinthecontrolreactionwithoutthecharacteristicbrightbandsmaybeindicativeofPCRovercycling(seeTroubleshootingGuide,SectionX.B).Ifthecharacteristicbandsarepresentbutweak,thismaybeindicativeofPCRundercycling(seeTroubleshootingGuide,SectionX.B).ThenumberandpositionofthebandsyouobtainwithyourexperimentalRNAmaydifferfromthoseshownforthecontrolreaction.Furthermore,cDNApreparedfromsomemammalian tissuesources (e.g., humanbrain, spleen,andthymus)maynotdisplayanybrightbands,duetotheveryhighcomplexityofthepolyA+RNA.



Figure 4. Analysis of ds cDNA synthesized for library construction. 1µl(1.0µg)oftheControlHumanPlacentalTotalRNAprovidedinthekitwasusedasstartingmaterialinafirst-strandcDNAsynthesis.2µlofthesscDNAthenservedastemplateforLDPCR-basedsecond-strandsynthesisusing15thermalcycles,accordingtotheprotocolinSectionV.A5-µlsampleofthePCRproduct(i.e.,dscDNA)waselectrophoresedona1.1%agarose/EtBrgel.LaneM:1-kbDNAladdersizemarkers,0.1µgloaded.Thearrowindicatesthestrongbandat900bptypicallyseenforhumanplacentaltotalRNA.

VI. Analysis of Results for Library Construction continued

M dscDNA

←

kb

126543

21.6

1

0.5



VII. SMART cDNA Synthesis Protocol

Important: This protocol is designed for synthesizing SMART cDNA forapplications other than library construction, such as Clontech PCR-Select™cDNA Subtraction or Virtual Northern Blots (See Appendix). To synthesizeSMARTcDNAforlibraryconstruction,usetheprotocolinSectionsVandVI.

Figure 5. Protocol guide for SMART cDNA synthesis for PCR-Select cDNA subtraction and other applications.


Virtual Northerns* (Appendix) and probes

First-strand ss cDNA(Section VII.A)

SMART ds cDNA(Section VII.B)

Optimization of PCR cycles

Agarose/EtBr gel analysis(Compare to Figure 7)

Troubleshooting(Section X.C)

Column chromatography(Section VIII.A)

Rsa I digestion†

(Section VIII.B)

Purification*(Section VIII.C)

Clontech PCR-Select™ cDNA subtraction†

*Reagents for these procedures are included in the SMART PCR cDNA Synthesis Kit.

† Reagents for these procedures are included in the Clontech PCR-Select cDNA Subtraction Kit.



VII. SMART cDNA Synthesis Protocol continued

Important: If you are planning to proceed with the Clontech PCR-SelectcDNASubtractionprotocol,werecommendreadingtheUserManualforcDNASubtraction before proceeding with first strand cDNA synthesis using theSMARTmethod.ThecDNASubtractionKitsuppliesadifferentRNAcontrolthatshouldbeusedtosynthesizecDNAaccordingtothePCR-SelectUserManual(whichdescribesanon-SMARTmethod).Inaddition,usethecontrolprovidedin thiskit to troubleshootanyproblemsusing theSMARTprotocol.Formoreinformationaboutusingthesecontrols,seeSectionVIII.DofthisUserManual.

A. First-Strand cDNA Synthesis 1.ForeachsampleandtheControlHumanPlacentalTotalRNA,combine

thefollowingreagentsinasterile0.5-mlreactiontube:

1–3µl RNAsample* (0.025–1µgofpolyA+or0.05–1µgoftotalRNA) 1µl 3'SMARTCDSPrimerIIA(12µM) 1µl SMARTIIAOligonucleotide(12µM) xµl DeionizedH2O

5µl Totalvolume*Forthecontrolsynthesis,add1µl(1µg/µl)ofControlHumanPlacentalTotalRNA.

2.Mixcontentsandspinthetubebrieflyinamicrocentrifuge. 3. Incubatethetubeat70°Cinathermalcyclerfor2min. 4.Spin the tube briefly in a microcentrifuge to collect contents at the

bottom.Keeptubeatroomtemperature. 5.Addthefollowingtoeachreactiontube:

2µl 5XFirst-StrandBuffer 1µl DTT(20mM) 1µl dNTPMix(10mMofeachdNTP) 1µl PowerScriptReverseTranscriptase 6.Gentlyvortexandspinthetubesbrieflyinamicrocentrifuge. 7. Incubatethetubesat42°Cfor1hrinanairincubator.

Note:Ifyouuseawaterbathorthermalcyclerforthisincubation,coverthereactionmixturewithonedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

8.Dilutethefirst-strandreactionproductbyaddingtheappropriatevolumeofTEbuffer(10mMTris[pH7.6],1mMEDTA):

• Add40µlofTEbufferifyouusedtotalRNAasstartingmaterial. • Add450µlofTEbufferifyouusedmorethan0.2µgofpolyA+RNA

asstartingmaterial. • Add90µlofTEbufferifyouusedlessthan0.2µgofpolyA+RNA

asstartingmaterial.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 �0 Version No. PR6Z2173



9.Heattubesat72°Cfor7min. 10.Samplescanbestoredat–20°Cforuptothreemonths.

B. cDNA Amplification by LD PCR Table II providesguidelines foroptimizingyourPCR,dependingon the

amountoftotalorpolyA+RNAusedinthefirst-strandsynthesis.TheseguidelinesweredeterminedusingtheControlHumanPlacentalTotalRNAandanauthorizedhot-lidthermalcycler;optimalparametersmayvarywithdifferenttemplatesandthermalcyclers.Todeterminetheoptimalnumberofcyclesforyoursampleandconditions,westronglyrecommendthatyouperformarangeofcycles:15,18,21,and24cycles(Figure6).

Figure 6. Optimizing PCR parameters for SMART™ cDNA synthesis. NotethatforsamplesnotusedforcDNAsubtraction,youwillonlyhavetwotubespersampleorcontrol:oneexperimentalsampletubeandone“extra”tube.

“extra” tube

15 PCR cycles

store at 4°C

3 PCR cycles

3 PCR cycles

3 PCR cycles

First-strand ss cDNA(from Section VII.A)

two tubes for PCR-Select samples

Run aliquots on a 1.2% agarose/EtBr gel

remove aliquot

remove aliquot

remove aliquot

remove aliquot

Determine optimal number of PCR cycles(Section VIII, Figure 7)

Run additional PCR cyclesto achieve optimal number

Protocol No. PT3041-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2173 �1



Foreachsampleandcontrol,setupanextrareactiontubetodeterminetheoptimalnumberofPCRcycles.IfyouplantouseyourSMARTcDNAforClontechPCR-SelectcDNAsubtraction,youshouldsetupatotalofthreetubesforeachtesteranddriversample(Figure6).Inourexperience,each100-µlreactiontypicallyyields1–3µgofdscDNAafterthePCRandpurificationsteps(SectionVIII).Subtractionusuallyrequires2µgofdrivercDNA,sotwotubesofSMARTcDNAshouldbesufficient;twotubeswillalsobeampleforthetester.ToensurethatyouhavesufficientcDNA,youshouldestimatetheyieldofSMARTcDNAbyUVspectrophotometry.

1.Preheatathermalcyclerto95°C. 2.Foreachreaction,aliquottheappropriatevolume(seeTableII,below)

ofeachdilutedcDNAintoalabeled0.5-mlreactiontube.Ifnecessary,adddeionizedH2Otoadjustthevolumeto10µl.

tableii:guidelinesforsettinguppcr

Total RNA Volume of diluted Typical optimal (µg) ss cDNA* for PCR (µl) No. of PCR cycles

~1.0 1µl 17–19

~0.5 2µl 17–19

~0.25 4µl 17–19

~0.1 10µl 17–19

~0.05 10µl 19–21

Poly A+ RNA Volume of diluted Typical optimal (µg) ss cDNA* for PCR (µl) No. of PCR cycles

~1.0 1µl 16–18

~0.5 2µl 16–18

~0.1–0.25 4µl 16–18

~0.05 8µl 16–18

~0.025 10µl 17–19 *FromStepVII.A.10.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 �� Version No. PR6Z2173


3.Prepare a Master Mix for all reaction tubes, plus one additionaltube.Combinethefollowingcomponentsintheordershown:

perrxn 74µl DeionizedH2O 10µl 10XAdvantage2PCRBuffer 2µl 50XdNTP(10mMofeachdNTP) 2µl 5'PCRPrimerIIA(12µM) 2µl 50XAdvantage2PolymeraseMix

90µl Totalvolume

4.Mixwellbyvortexingandspinthetubebrieflyinamicrocentrifuge. 5.Aliquot90µlofthePCRMasterMixintoeachtubefromStep2. 6.Capthetube,andplaceitinthepreheatedthermalcycler.Ifnecessary,

overlaythereactionmixturewithtwodropsofmineraloil. 7.Commencethermalcyclingusingthefollowingprogram: •95°C 1min •xcycles*: 95°C 15sec 65°C 30sec 68°C 6min

*ConsultTableIIforguidelines.Subject all tubes to 15 cycles.Then,usetheextratubeforeachreactiontodeterminetheoptimalnumberofPCRcycles,asdescribedinStep8(below).Storetheothertubesat4°C.

8.ForeachextraPCRtube,determinetheoptimalnumberofPCRcycles(seeFigure6):

a. Transfer15µl fromthe15-cyclePCRtoacleanmicrocentrifugetube(foragarose/EtBrgelanalysis).

b. Runthreeadditionalcycles(fora totalof18)with theremaining85µlofthePCRmixture.

c. Transfer15µl fromthe18-cyclePCRtoacleanmicrocentrifugetube(foragarose/EtBrgelanalysis).

d. Runthreeadditionalcycles(fora totalof21)with theremaining70µlofPCRmixture.

e. Transfer15µl fromthe21-cyclePCRtoacleanmicrocentrifugetube(foragarose/EtBrgelanalysis).

f. Runthreeadditionalcycles(fora totalof24)with theremaining55µlofPCRmixture.

9.Electrophorese5µlofeachaliquotofeachPCRreactionalongside0.1µgof1-kbDNAsizemarkerona1.2%agarose/EtBrgel in1X


Protocol No. PT3041-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2173 �3


TAEbuffer.Determinetheoptimalnumberofcyclesrequiredforeachexperimentalandcontrolsample(seeFigure7,SectionIX).

10.Retrievethe15-cyclePCRtubesfrom4°C,returnthemtothethermalcycler,andsubject themtoadditionalcycles, ifnecessary,untilyoureachtheoptimalnumber.

11.Whenthecyclingiscompleted,analyzea5-µlsampleofeachPCRproductalongside0.1µgof1-kbDNAsizemarkerona1.2%agarose/EtBrgelin1XTAEbuffer.CompareyourresultstoFigure7toconfirmthatyourreactionsweresuccessful.

12.Add2µlof0.5MEDTAtoeachtubetoterminatethereaction. 13.Transfer7µlofyourrawPCRproducttoacleanmicrocentrifugetube

andlabelthistube“SampleA”.Storeat–20°C.YouwilluseSampleAforanalysisofcolumnchromatography,asdescribedinSectionIX.B.

YounowhaveSMARTdscDNAreadytouseforapplicationssuchasVirtualNorthernblottingorgenerationof cDNAprobes.ForPCR-Select cDNAsubtraction,proceedwiththefollowingprotocol(StepVIII.A,below).

VIII. Protocol for Clontech PCR-Select™ cDNA Subtraction

A. Column Chromatography(PCR-SelectUsersonly!) 1.Foreveryexperimentalsampleandcontrol,combinethetworeaction

tubesofPCRproduct(fromSectionVII.B)intoa1.5-mlmicrocentrifugetube.

2.Addanequalvolumeofphenol:choloroform:isoamylalcohol(25:24:1).Vortexthoroughly.

3.Centrifuge the tubes at 14,000 rpm for 10 min to separate thephases.

4.Removethetop(aqueous)layerandplaceitinaclean1.5-mltube. 5.Add700 µl of n-butanol and vortex the mixture thoroughly.Butanol

extractionallowsyoutoconcentrateyourPCRproducttoavolumeof40–70µl.Note: Addition of too much n-butanol may remove all the water and precipitate thenucleicacid.Ifthishappens,addwatertothetubeandvortexuntilanaqueousphasereappears.

6.Centrifugethesolutionatroomtemperatureat14,000rpmfor1min. 7.Removeanddiscardtheupper(n-butanolorganic)phase. 8. Ifyoudonotendupwithavolumeof40–70µl,repeatsteps5–7.

Note: If your volume is <40 µl, add H2O to the aqueous phase to adjust volume to40–70µl.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 �4 Version No. PR6Z2173


9. Invert a CHROMA SPIN 1000 Column several times to completelyresuspendthegelmatrix.Note:Checkforairbubblesinthecolumnmatrix.Ifbubblesarevisible,resuspendthematrixinthecolumnbufferbyinvertingthecolumnagain.

10.Remove the topcap from thecolumn,and then remove thebottomcap.

11.Place the column into a 1.5-ml centrifuge tube or a 17 x 100 mmtube.

12.Discardanycolumnbuffer that immediatelycollects in thetubeandadd1.5mlof1XTNEbuffertothecolumn.

13.Letthebufferdrainthroughthecolumnbygravityflowuntilyoucanseethesurfaceofthegelbeadsinthecolumnmatrix.Thetopofthecolumnmatrixshouldbeatthe0.75-mlmarkonthewallofthecolumn.Ifyourcolumncontainsmuchlessmatrix,discarditanduseanothercolumn.

14.Discardthecollectedbufferandproceedwithpurification. 15.Carefullyandslowlyapplythesampletothecenterofthegelbed’s

flatsurface.Donotallowanysampletoflowalongtheinnerwallofthecolumn.

16.Apply25µlof1XTNEbufferandallowthebuffertocompletelydrainoutofthecolumn.

17.Apply150µlof1XTNEbufferandallowthebuffertocompletelydrainoutofthecolumn.

18.Transfercolumntoaclean1.5-mlmicrocentrifugetube. 19.Apply320µlof1XTNEbufferandcollecttheeluateasyourpurified

dscDNAfraction.Transfer10µlofthisfractiontoacleanmicrocentrifugetubeandlabelthistube“SampleB”.Storeat–20°C.Usethisaliquotforagarose/EtBrgelanalysis(Step21,below).

20.Apply75µlof1XTNEbufferandcollecttheeluateinacleanmicro-centrifugetube.Labelthistube“SampleC”andstoreat–20°C.Save this fraction until after you perform agarose/EtBr gel analysis(Step21,below).

21.ToconfirmthatyourPCRproductispresentinthepurifieddscDNAfraction, perform the agarose/EtBr gel analysis as described inSectionIX.B.

B. Rsa I Digestion (PCR-SelectUsersonly!) Thisstepgeneratesshorter,blunt-endeddscDNAfragments,whichare

necessaryforbothadaptorligationandsubtraction.

BeforeproceedingwithRsaIdigestion,setasideanother10µlofpurified

VIII. Protocol for Clontech PCR-Select... continued

Protocol No. PT3041-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2173 ��


dscDNAforagarose/EtBrgelanalysistoestimatethesizerangeofthedscDNAproducts(Step4,below).Labelthistube“SampleD”.

1.AddthefollowingreagentstothepurifiedcDNAfractioncollectedfromtheCHROMASPINColumn(StepVIII.A.21):

10XRsaIrestrictionbuffer 36µlRsa I (10units) 1.5µl

2.Mixbyvortexingandspinbrieflyinamicrocentrifuge. 3. Incubateat37°Cfor3hr. 4.ToconfirmthatRsaIdigestionwassuccessful,electrophorese10µl

ofuncutdscDNA(SampleD)and10µlofRsaI-digestedcDNAona1.2%agarose/EtBrgelin1XTAEbuffer(seeSectionIX.CinthisUserManualandSectionV.BintheClontechPCR-SelectUserManual).

5.Add8µlof0.5MEDTAtoterminatethereaction. 6.Transfer10µlofthedigestedcDNAtoacleanmicrocentrifugetube,

labelthistube“SampleE”,andstoreat–20°C.YouwillcomparethissampletothePCRproductafterfinalpurification,asdescribedinSec-tionIX.D.

C. Purification of Digested cDNA (PCR-SelectUsersonly!) YoumaypurifyyourdigestedcDNAusinganysilicamatrix-basedPCRpu-

rificationsystem,suchasthoseofferedbyClontech(seeRelatedProducts,SectionXII).Alternatively,aphenol:chloroformextractionmaybeperformed;however,thismaydecreasetheefficiencyofthecDNAsubtraction.Thefol-lowingpurificationprocedurehasbeenoptimizedusingSMARTdscDNAandtheNucleoTrapPCRKit(Cat.No.636020;notincludedwithPCR-SelectKit).

Before you start:Add64mlof95%ethanoltotheBufferNT3forafinalconcentrationofapproximately85%.TheappropriatevolumeisalsolistedontheBufferNT3bottle.

1.AliquottheRsaI-digestedcDNA(SectionVIII.B.6,above)intotwoclean,1.5-mlmicrocentrifugetubes(approximately170µlineachtube).

2.VortextheNucleoTrapSuspensionthoroughlyuntilthebeadsarecom-pletelyresuspended.

3.Add680µlofBufferNT2and17µlofNucleoTrapSuspensiontoeachtubeofdigestionmixture.

4. Incubatethesampleatroomtemperaturefor10min.Mixgentlyevery2–3minduringtheincubationperiod.

5.Centrifugethesampleat10,000xgfor1minatroomtemperature.Discardthesupernatant.





6.Add680µlofBufferNT2tothepellet.Mixgentlytoresuspend.Centrifugeat10,000xgfor1minatroomtemperature.Removethesupernatantcompletelyanddiscard.

7.Add680µlofBufferNT3tothepellet.Mixgentlytoresuspend.Centri-fugethesampleat10,000xgfor1minatroomtemperature.Removethesupernatantcompletelyanddiscard.

8.RepeatStep7. 9.Centrifugethepelletagainat10,000xgfor1minatroomtemperature.

Airdrythepelletfor15minatroomtemperature(orat37°Ctospeedupevaporation).Note: Donotuseaspeedvactodrythepellet;speedvacstendtooverdrythebeads,whichleadstolowerrecoveryrates.

10.Add50µlofTEbuffer(pH8.0)tothepellet.Resuspendthepelletbymixing gently. Combine the resuspended pellets into one tube. Mixgently.

11.ElutetheDNAbyincubatingthesampleat50°Cfor5min.Gentlymixthesuspension2–3timesduringthisincubationstep.

12.Centrifugethesampleat10,000xgfor30secatroomtemperature.Transferthesupernatant,containingthepureDNAfragment,toaclean1.5-mlmicrocentrifugetube.Note:RepeatingSteps10–12canincreaseyieldsapproximately10–15%.

13.Applythesupernatanttoamicrofiltrationcolumnthathasbeeninsertedintoa1.5-mltube.Centrifugefor5minanddiscardthecolumn.

14.Transfer 6 µl of the filtered DNA solution to a clean 1.5-ml micro-centrifuge tube containing 14 µl of deionized H2O. Label this tube“SampleF”andstoreat–20°C.YouwillusethissampletoanalyzetheSMARTcDNAafterpurification,asdescribedinSectionIX.D.

15.Toprecipitate theDNA,add1/2volumeof4Mammoniumacetate(e.g.,50µlfora100-µlsample),thenadd2.5volumesof95%ethanol(e.g.,375µlfor150µlsample+ammoniumacetate)totheremainingsamplefromStep14.

16.Vortexthemixthoroughlyandcentrifugethetubesat14,000rpmfor20minatroomtemperature.

17.Carefullyremoveanddiscardthesupernatant. 18.Overlaythepelletwith500µlof80%ethanol. 19.Centrifuge the tubeat14,000 rpmfor10min.Carefully remove the

supernatantanddiscard. 20.Airdrythepelletsfor5–10min. 21.Dissolvethepelletin6.7µlof1XTNEbuffer.




22.Transfer 1.2 µl to a clean 1.5-ml microcentrifuge tube containing11µlofdeionizedH2O,labelthistube"SampleG,"andstorethere-mainingsampleat–20°C.Use10µlofthedilutedDNAtoassesstheyieldofDNAbyUVspectrophotometry.Foreachreaction,weusuallyobtain1–3µgofSMARTcDNAafterpurification.Fortwotubes,youshouldobtainatotalof2–6µgofcDNA.Ifyouryieldislowerthanthis,performtheagarose/EtBrgelanalysisdescribedinSectionIX.D.

23. IfyourDNAconcentrationis>300ng/µl,diluteyourcDNAtoafinalcon-centrationof300ng/µlin1XTNEbuffer,andfollowtheadaptorligationstepinaccordancewiththeClontechPCR-SelectcDNAsubtractionprotocol.

24.YourdigesteddscDNAisnowreadyforadaptorligation,asdescribedinSectionIV.FoftheUserManualfortheClontechPCR-SelectcDNASubtractionKit(Cat.No.637401).BesuretoreadSectionVIII.DbelowforimportantcDNAsubtractioncontrolprocedures.

D. Controls for Clontech PCR-Select™ cDNA Subtraction Westronglyrecommendthatyouperformthefollowingcontrolsubtractions.

PleaserefertoSectionIVofthePCR-SelectUserManual.1. Control subtractionusing thehuman skeletal muscle poly A+ RNA

(includedinthePCR-SelectKit)Use the conventional method (as described in the PCR-Select UserManual)tosynthesizedscDNAfromthecontrolhumanskeletalmusclepoly A+ RNA provided in the PCR-Select Kit.Then, set up a “mock”subtraction:useaportionofthehumanskeletalmusclecDNAasthedriver,andmixanotherportionwithasmallamountofthecontrolHae III-digestedφX174DNAfromthePCR-SelectKitasthetester.Thiscontrolsubtraction,whichisdescribedindetailinthePCR-SelectUserManual,isthebestwaytoconfirmthatthemultistepsubtractionprocedureworksinyourhands.

2. Controlsubtractionusingthe human placental total RNA (includedintheSMARTkit)

UsetheSMARTkittoamplifytheControlHumanPlacentalTotalRNA;then,performamocksubtractionasdescribedforControlNo.1:useaportionofthehumanplacentalcDNAasthedriver,andmixanotherpor-tionwithasmallamountofthecontrolHae III-digestedφX174DNAfromthePCR-SelectKitasthetester.IfControlNo.1works,butControlNo.2doesnot,youmayassumethattheSMARTcDNAamplificationand/orpurificationfailed.Inthiscase,tryreducingthenumberofPCRcyclesfor thecDNAamplificationandtroubleshootyourpurificationprotocol(SectionVIII.C).



IX. Analysis for Clontech™ PCR-Select Subtraction

Figure7showsatypicalgelprofileofdscDNAsynthesizedusingtheControlHumanPlacentalTotalRNAand theSMARTprotocoloutlined inSectionVII.As indicatedby thearrow,youshouldobserveastrong,distinctbandat900bp. Ingeneral,cDNAsynthesized frommammalian totalRNAshouldappearona1.2%agarose/EtBrgelasamoderatelystrongsmearfrom0.5–6kbwithsomedistinctbands.ThenumberandpositionofthebandsyouobtainwillbedifferentforeachparticulartotalRNAused.Furthermore,cDNApreparedfromsomemammaliantissuesources(e.g.,humanbrain,spleen,andthymus)maynotdisplaybrightbandsduetotheveryhighcomplexityofthepolyA+RNA.Fornonmammalianspecies,thesizedistributionmaybesmaller(seeSectionX.A.2formoredetails).

A. Determining the Optimal Number of PCR Cycles (Step VII.B.8) Forbestresults,youmustoptimizethePCRcyclingparametersforyour

experiment,asdescribedinSectionVII.B(Figure6).ChoosingtheoptimalnumberofPCRcyclesensuresthatthedscDNAwillremainintheexponentialphaseofamplification.WhentheyieldofPCRproductsstopsincreasingwithmorecycles,thereactionhasreacheditsplateau.OvercycledcDNAisaverypoortemplateforcDNAsubtraction.Undercycling,ontheotherhand,resultsinaloweryieldofyourPCRproduct.Theoptimalnumberofcyclesforyourexperimentisonecyclefewerthanisneededtoreachtheplateau.Beconservative:when indoubt, it isbetter tousefewercyclesthantoomany.

Figure 7. Analysis for optimizing PCR parameters. 5µlofeachPCRproductwaselectrophoresedona1.2%agarose/EtBrgel in1XTAEbufferfollowingtheindicatednumberofPCRcycles.Theoptimumnumberofcyclesdeterminedinthisexperimentwas17.LaneM:1-kbDNAladdersizemarkers,0.1µgloaded.Thearrowindicatesthestrongbandat900bptypicallyseenforhumanplacentaltotalRNA.

TotalRNAPolyA+RNA

M1518212415182124cycleskb

1232

1.6

1

0.5

←



IX. Analysis for Clontech PCR-Select Subtraction... cont.

We have optimized the PCR cycling parameters presented in thisUser Manual using an authorized hot-lid thermal cycler and theAdvantage®2PCRKit(Cat.Nos.639206&639207).Theseparametersmayvarywithdifferentpolymerasemixes,templates,andthermalcyclers.WestronglyrecommendthatyouoptimizethenumberofPCRcycleswithyourexperimentalsample(s)andtheControlHumanPlacentalTotalRNA.Trydifferentnumbersofcycles;then,analyzeyourresultsbyelectrophoresing5µlofeachproductona1.2%agarose/EtBrgelin1XTAEbuffer.

Figure7providesanexampleofhowyouranalysisshouldproceed.Inthisexperiment,thePCRreacheditsplateauafter18cycles;thatis,theyieldofPCRproductsstoppedincreasing.After21and24cycles,asmearap-pearedinthehighmolecularweightregionofthegel,indicatingthatthereactionwasovercycled.Becausetheplateauwasreachedafter18cycles,theoptimalnumberofcyclesforthisexperimentwouldbe17.

B. Column Chromatography (Section VIII.A) ToanalyzethedscDNAaftercolumnchromatography,electrophorese3µl

oftheunpurifiedPCRproduct(SampleA,fromSectionVII.B.13)alongside10µlofthePCRproductpurifiedbycolumnchromatography(SampleB,fromSectionVIII.A.19)and10µlofthesecondfraction(SampleC,fromSectionVIII.A.20)ona1.2%agarose/EtBrgel.Compare the intensitiesofSampleAandSampleB,andestimatethepercentageofPCRproductthatremainsaftercolumnchromatography.TheyieldofcDNAaftercolumnchromatographyistypically50percent.Ifyouryieldis<30percent,checktoseeifitispresentinthesecondfraction,SampleC.Ifthissecondfrac-tionhasahigheryieldofcDNAthanthefirst,combinethefractionsandproceedwithSectionVIII.B.OtherwiseifthecDNAisnotpresentinSampleC,repeatthePCRandcolumnchromatographysteps.

C. Rsa I Digestion (Section VIII.B) ToconfirmthatRsaIdigestionwassuccessful,electrophorese10µlofuncut

dscDNA(SampleD,fromSectionVIII.B)alongside10µlofRsaI-digestedcDNA(fromSectionVIII.B.4)ona1.2%agarose/EtBrgel.Comparetheprofilesofbothsamples.BeforeRsaIdigestion,dscDNAshouldappearasasmearfrom0.5–10kbwithbrightbandscorrespondingtoabundantmRNAs.(ForsomeRNAsamplesfromnonmammalianspecies,thesizedistributionmaybeonly0.5–3kb.)AfterRsaIdigestion,thesmearshouldrangefrom0.1–2kb.ThisresultwillbesimilartothatshownintheUserManualforthePCR-SelectKit.



D. Purification of Digested cDNA (Section VIII.C) ToanalyzetheyieldofpurifiedSMARTcDNA,electrophorese10µlofRsa

I-digestedcDNAbeforepurification(SampleE,fromStepVIII.B.6)alongside10µlofpurifieddilutedcDNAbeforeethanolprecipitation(SampleF,StepVIII.C.14)and1.8µlofpurifieddilutedcDNAafterethanolprecipitation(SampleG,fromStepVIII.C.22)ona1.5%agarose/EtBrgel.ComparetheintensitiesofthesamplesandestimatewhatpercentageofRsaI-digestedPCRproductremainsafterpurificationandethanolprecipitation.TheyieldofcDNAafterpurificationusingtheNucleoTrapPCRKitandethanolpre-cipitationistypically70percent.Ifyouryieldis<30percent,troubleshootyourpurificationprotocolorconsultthetroubleshootingguideoftheUserManualforthatparticularpurificationkit.

IX. Analysis for Clontech PCR-Select Subtraction... cont.



A. First-Strand cDNA Synthesis and SMART PCR Amplification (Sections V.A–B & VII.A–B)

1.Lowmolecularweight(sizedistribution<3kb),pooryield,ornoPCRproductobservedfortheControlHumanPlacentalTotalRNA

a. Proper template switching, which is essential to the SMARTtechnology,requirestheuseofanMMLVRNaseH–pointmutantreversetranscriptasesuchasPowerScriptReverseTranscriptase(includedwitheachSMARTKit).

b. RNAsmayhavedegradedduringstorageand/orfirst-strandsynthesis.PoorqualityRNAstartingmaterialwillreducetheabilitytoobtainfull-lengthcDNAs.RNAmustbestoredat–70°C.Yourworkingarea,equipment,andsolutionsmustbefreeofcontaminationbyRNaseA.

c. Youmayhavemadeanerrorduringtheprocedure,suchasusinga suboptimal incubation temperature or omitting an essentialcomponent.Carefullychecktheprotocolandrepeatthefirst-strandsynthesisandPCR.

d. TheconditionsandparametersforPCRmayhavebeensuboptimal.TheoptimalnumberofPCRcyclesmayvarywithdifferentPCRmachines,polymerasemixes,orRNAsamples.IfyourPCRreachesitsplateauafter24cyclesormore,theconditionsofyourPCRmaynotbeoptimal.ChecktheprotocolandrepeatthePCRusingafresh2-µlaliquotofthefirst-strandproduct.

2.PooryieldortruncatedPCRproductfromyourexperimentalRNA IfthereactionwiththeControlHumanPlacentalTotalRNAwassuccessful,

butyourexperimentfailed,yourexperimentalRNAsamplemaybetoodiluteordegraded,ormaycontain impurities that inhibit first-strandsynthesis.IfyourRNAsamplewaspreparedfromanonmammalianspecies,theapparentlytruncatedPCRproductmayactuallyhavethenormalsizedistributionforthatspecies.Forexample,forinsects,thenormalRNAsizedistributionmaybe<2–3kb.Ifyouhavenotalreadydoneso,electrophoreseasampleofyourRNAona formaldehyde/agarose/EtBrgeltodetermineitsconcentrationandanalyzeitsquality(seeSectionIVformoredetails).

a. TheconcentrationofyourexperimentalRNAislow,butthequalityisgood.

RepeattheexperimentusingmoreRNAand/ormorePCRcycles. b. YourexperimentalRNAhasbeenpartiallydegraded(bycontaminating

RNases)beforeorduringfirst-strandsynthesis.

X. Troubleshooting Guide



Repeat the experiment using a fresh lot or preparation of RNA.CheckthestabilityofyourRNAbyincubatingasmallsamplefor2hrat42°C.Then,electrophoreseitonaformaldehyde/agarose/EtBrgelalongsideanunincubatedsample.IftheRNAisdegradedduringincubation,itwillnotyieldgoodresultsinthefirst-strandsynthesis.Inthiscase,re-isolatetheRNAusingadifferenttechnique,suchasoneemployedbyourRNAisolationkits(seeRelatedProductsfororderinginformation).Severaladditionalroundsofphenol:chloroformextractionmaydramaticallyincreaseRNAstability.

c. YourexperimentalRNAsamplecontainsimpuritiesthatinhibitcDNAsynthesis.

In some cases, ethanol precipitation of your existing total RNA,followedbywashingtwice in80%EtOH,mayremove impurities.Ifthisfails,reisolatetheRNAusingadifferenttechnique,suchasoneemployedbyourRNAisolationkits(seeRelatedProductsfororderinginformation).

B. Special Considerations for Library Construction (Sections V & VI) 1.LowyieldofPCRproduct a. Too few thermal cycleswereused in thePCRstep.Another in-

dicationofPCRundercyclingisacDNAsizedistribution<3kbifthemRNAsourcewasmammalian. (Forsomesources,suchasmany insectspecies, thenormalmRNAsizedistributionmaybe<2–3kb.)Ifyoususpectthatundercyclingistheproblem,incubatethePCRmixturefortwomorecyclesandrechecktheproduct.IfyoualreadyusedthemaximumrecommendednumberofcyclesindicatedinTableI,increasebythreemorecycles.IfincreasingthenumberofcyclesdoesnotimprovetheyieldofPCRproduct,repeatthePCRusingafresh2-µlaliquotofthefirst-strandproduct.

b. IfyoustillobtainalowyieldofPCRproduct,itmaybeduetoalowyieldoffirst-strandcDNA.Possibleproblemswiththefirst-strandreactionincludeamistakeintheprocedure(suchasusingasubop-timalincubationtemperatureoromittingacomponent)ornotusingenoughRNAinthereaction.ItisalsopossiblethattheRNAhasbeenpartiallydegraded(bycontaminatingRNases)beforeorduringthefirst-strandsynthesis.Reminder:problemswiththefirst-strandcDNAsynthesiscanbemoreeasilydiagnosedifyouperformparallelreactionsusingtheControlRNAprovidedinthekit.IfgoodresultswereobtainedwiththeControlRNAbutnotwithyourexperimentalRNA,thentheremaybeaproblemwithyourRNA.

Theeasiestway to check thequalityof thefirst-strandcDNA isbyusingasmall sampleof it asaPCR templatewith3' and5'gene-specificprimers,suchasHumanβ-ActinControlAmplimers(Cat. No. 639001, Cat. No. 639002). If the first-strand synthesis

X. Troubleshooting Guide continued



hasbeensuccessful,aPCRproductoftheexpectedsizewillbegenerated.

2.NobrightbandsdistinguishableinthePCRproduct For most mammalian RNA sources, there should be several bright

bandsdistinguishableagainstthebackgroundsmearwhenasampleofthePCRproductisrunonagel.Ifbrightbandsareexpectedbutarenotvisible,andthebackgroundsmear isvery intense,youmayhaveovercycledyourPCR.Ifyoususpectthatyourproblemisduetoovercycling,thenthePCRstep(SectionV.B)mustberepeatedwithafresh2-µlsampleoffirst-strandcDNA,using2–3fewercycles.

C. Preparation for Clontech PCR-Select™ cDNA Subtraction (Sections VII–IX)

FortroubleshootingtheactualPCR-Selectsubtractionprocedure,pleaserefertotheUserManualfortheClontechPCR-Select™cDNASubtractionKit.Here,weprovideatroubleshootingguideforpreparingSMARTcDNAforsubtraction(describedinSectionVIIandVIII).

1.LowyieldofcDNAaftercolumnchromatography(SectionVIII.A) Possiblereasonsforlowyieldincludethefollowing: a. You may have applied the wrong volume of buffer to the

CHROMASPINcolumn,orcollectedthewrongvolumeofbufferfromthecolumn.Carefullychecktheprotocolandrepeatcolumnchromatography.

b. Yourcolumnmayhaveleakedduringshipping.Ifyourcolumncontainslessthan750µlofmatrix,discarditanduseanothercolumn.

2.FailureofRsaIdigestion(SectionVIII.B) Ifthesizedistributionofyoursampleand/orcontrolcDNAisnotreduced

afterRsaIdigestion,checktherecipeforTNEbuffer.IfyouusedthecorrectrecipeforTNEbuffer,performphenol:chloroformextractionandethanolprecipitation;then,repeattheRsaIdigestion.

3.LowyieldofcDNAafterpurificationofdigestedcDNA(SectionVIII.C)

Possiblereasonsforlowyieldincludethefollowing: a. Loss of cDNA during purification.Troubleshoot your purification

procedure. b. LossofcDNAduringethanolprecipitation.Checkthevolumesof

theammoniumacetateandethanol.Repeatpurificationandethanolprecipitation.

c. YourPCRdidnotreachtheplateau(i.e.,thereactionwasunder-cycled).PerformmorePCRcycles.OptimizethenumberofcyclesasdescribedinSectionIX.

X. Troubleshooting Guide continued



XI. References

Barnes,W.M.(1994)PCRamplificationofupto35-kbDNAwithhighfidelityandhighyieldfromλbacteriophagetemplates.Proc. Natl. Acad. Sci. USA91:2216–2220.

ChenchikA.,Moqadam,F.&Siebert,P.(January1995)MarathoncDNAamplification:Anewmethodforcloningfull-lengthcDNAs.Clontechniques X(1):5–8.

Chenchik,A.,Moqadam,F.&Siebert,P.(1996)Anewmethodforfull-lengthcDNAcloningbyPCR.InA Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Ed.Krieg,P.A.(Wiley-Liss,Inc.),pp.273–321.

Chenchik,A.,Zhu,Y.Y.,Diatchenko,L.,Li,R.,Hill,J.&Siebert,P.D.(1998)Generationanduseofhigh-qualitycDNAfromsmallamountsoftotalRNAbySMARTPCR.InGene Cloning and Analysis by RT-PCR(BioTechniquesBooks,MA),pp.305–319.

Chomczynski,P.&Sacchi,N.(1987)Single-stepmethodofRNAisolationbyacidguanidiniumthio-cyanate-phenol-chloroformextraction.Anal. Biochem.162:156–159.

Diatchenko,L.,Lau,Y.-F.C.,Campbell,A.P.,Chenchik,A.,Moqadam,F.,Huang,B.,Lukyanov,S.,Lukyanov,K.,Gurskaya,N.,Sverdlov,E.D.&Siebert,P.D.(1996)Suppressionsubtractivehybridiza-tion:Amethodforgeneratingdifferentiallyregulatedortissue-specificcDNAprobesandlibraries.Proc. Natl. Acad. Sci. USA 93:6025–6030.

Endege,W.O.,Steinmann,K.E.,Boardman,L.A.,Thibodeau,S.N.&Schlegel,R.(1999)Represen-tativecDNAlibrariesandtheirutilityingeneexpressionprofiling.BioTechniques 26:542–550.

Farrell,Jr.,R.E.(1993)RNA Methodologies—A Lab Guide for Isolation and Characterization(Aca-demicPress,SanDiego,CA).

Gurskaya,N.G.,Diatchenko,L.,Chenchik,A.,Siebert,P.D.,Khaspekov,G.L.,Lukyanov,K.A.,Vagner,L.L.,Ermolaeva,O.D.,Lukyanov,S.A.&Sverdlov,E.D.(1996)EqualizingcDNAsubtractionbasedonselectivesuppressionofpolymerasechainreaction:CloningofJurkatcelltranscriptsinducedbyphytohemaglutininandphorbol12-myristate13-acetate.Anal. Biochem.240:90–97.

Kellogg, D. E., Rybalkin, I., Chen, S., Mukhamedova, N., Vlasik, T., Sieber t, P.& Chenchik, A. (1994)TaqStart Antibody: Hotstart PCR facilitated by a neutralizing monoclonalantibodydirectedagainstTaqDNApolymerase.BioTechniques16:1134–1137.

Lukyanov,S.A.,Gurskaya,N.G.,Tarabykin,V.S.&Sverdlov,E.D.(1994)HighlyefficientsubtractivehybridizationofcDNA.Biorganic Chem. (Russian)20:701–704.

Matz,M.,Lukyanov,S.,Bogdanova,E.,Diatchenko,L.,&Chenchik,A.(1999)AmplificationofcDNAendsbasedontemplate-switchingeffectandstep-outPCR.Nucleic Acids Res.27:1558–1560.

Morgan,J.G.,Dolganov,G.M.,Robbins,S.E.,Hinton,L.M.&Lovett,M.(1992)SelectiveisolationofnovelcDNAsencodedbytheregionsurroundingthehumanIL-4and-5genes.Nucleic Acids Res.20:5173–5179.

Pietu, G., Alibert, O., Guichard, V., Lamy, B., Bois, F., Leroy, E., Mariage-Sampson, R., Houl-gatte,R.,Soularue,P.&Auffray,C.(1996)Novelgenetranscriptspreferentiallyexpressedinhu-manmuscles revealedbyquantitativehybridizationofahighdensitycDNAarray.Genome Res. 6:492–503.

Sambrook,J.,&Russell,D.W.(2001)MolecularCloning:ALaboratoryManual,(ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY).

Siebert,P.D.,Chenchik,A.,Kellogg,D.E.,Lukyanov,K.A.&Lukyanov,S.A.(1995)AnimprovedmethodforwalkinginunclonedgenomicDNA.Nucleic Acids Res.23:1087–1088.

SMARTRACEcDNAAmplificationKit(January1999)ClontechniquesXIV(1):4–6.

Zhu,Y.Y.,Machleder,E.M.,Chenchik,A.,Li,R.&Siebert,P.M.(2001)Reversetranscriptasetem-plate switching: A SMARTTM approach for full-length cDNA library construction. BioTechniques30:892–897.



XII. Related Products

ForacompletelistingofallClontechproducts,pleasevisitwww.clontech.com

Cat. No.

• ClontechPCR-Select™cDNA SubtractionKit 637401

• ClontechPCR-Select™Differential 637403 ScreeningKit

• SMART™cDNALibraryConstructionKit 634901

• SMART™RACEcDNAAmplificationKit 634914

• Advantage®2PCRKit 639206 639207

• Advantage®2PolymeraseMix 639201 639202

• SprintAdvantage®SingleShots 639553 639554 639556

• SprintAdvantage®96Plate 639550

• PowerScript™ReverseTranscriptase 639500 639501

• NucleoTrap®mRNAMiniPurificationKit 636022

• NucleoSpin®RNAIIKit 635990

• PremiumTotalRNAs many

• PremiumPolyA+RNAs many

• TaqStart™Antibody 639250

• AmplimerSets many

• Marathon®-ReadycDNAs many

• MTN®MultipleTissueNorthernBlots many



After cloning your subtracted cDNA fragments, you should confirm that theyrepresent differentially expressed genes. Typically, this is accomplished byhybridizationtoNorthernblotsofthesameRNAsamplesusedasdriverandtesterforsubtraction.If,however,youhavelimitedsamplematerial,youmaywishtouseVirtualNorthernblotsforanalysis.ByusingthesameSMARTPCR-amplifiedtesteranddrivercDNAusedforsubtraction,youcanobtaininformationthatissimilartothatprovidedbystandardNorthernanalysis.EvenifacDNAdoesnotgiveasinglebandwhenhybridizedtoaVirtualNorthernblot,youcanstilldetectwhetherornotitisdifferentiallyexpressed.MultiplebandsonaVirtualNorthernblotmayresultfromdifferentcauses.ThecDNAmaybelongtoamulti-genefamily,ormaycontainanucleotiderepeat.Alternatively,atruncatedcopyofthegenemaybepresent.Todistinguishbetweenthesepossibilities,analysisshouldalsoincludeothermethods,suchasgenomicDNAsequencingorRACE.

To prepare a Virtual Northern blot, electrophorese your SMART PCR-amplifiedcDNA(beforepurification)onanagarose/EtBrgelanduseaSoutherntransferontoanylonmembrane(seeSambrooket al.,1989).AtClontech,weusetheTurboBlotterequipmentandprotocolfromSchleicher&Schuell.Figure8showshowVirtualNorthernblotscanbeusedtoconfirmdifferentialexpressionofsubtractedcDNAs.

Appendix: Virtual Northern Blots

Figure 8. Virtual Northern blot analysis of cDNA fragments expressed in cells producing γ-globin.ClontechPCR-SelectcDNAsubtractionwasperformedto isolatecDNAsthatwerepreferentiallyexpressedincellsproducingγ-globin.1µgoftotalRNAfromcellsproducingγ-globinwasusedasthetester;1µgoftotalRNAfromcellsproducingβ-globinwasusedasthedriver.TesteranddrivercDNAsweresynthesizedusingtheSMARTPCRcDNASynthesisKitandweresubjectedtoPCR-Selectsubtraction.84subtractedcDNAcloneswerearrayedonanylonmembranefordifferentialscreening.13ofthesesubtractedcDNAsshoweddifferentialsignalsandwerethereforecandidatesforfurtheranalysisbyVirtualNorthernblots.Differentialexpressionofall13cloneswasconfirmed;fourexamplesareshowninthisfigure.VirtualNorthernblotswerepreparedusingthesameSMARTPCR-amplifiedcDNAthatwasusedforsubtraction.Eachlanecontains0.5µgofSMARTcDNA.SubtractedcDNAfragments(γ-1,γ-2,γ-3,andγ-4)werelabeledwith[32P]-dCTPandhybridizedtotheVirtualNorthernblots.HybridizationwithG3PDHservesasacontrolforloading.Laneγ:Cellsproducingγ-globin.Laneβ:Cellsproducingβ-globin.

γ β γ β γ β γ β γ βkb

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Notes



Notes



Notice to Purchaser

Clontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,in vitrodiagnosticpurposes,therapeu-tics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.

SMART™TechnologyiscoveredbyU.S.PatentNos.5,962,271and5,962,272.For-ProfitandNot-For-ProfitpurchasersofSMART™Productsareentitledtousethereagentsforinternalresearch.However,thefollowingusesareexpresslyprohibited:(1)performingservicesforthirdparties;(2)identifyingnucleicacidsequencestobeincludedonnucleicacidarrays,blots,orinlibrariesorothercDNAcollectionswhicharethensoldtothirdparties.Reproduction,modifica-tion,reformulation,orresaleofthereagentsprovidedinSMART™Productsisnotpermitted.ForinformationonlicensingSMART™Technologyforcommercialpurposes,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

TurboBlotter™isatrademarkoftheWhatmanGroup.

NucleoTrap®andNucleoSpin®areregisteredtrademarksofMACHEREY-NAGELGmbH&Co.KG.

Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006

Notes

Documents

SMART PCR cDNA Synthesis Kit User Manual - genex.cl · PDF fileProtocol No. PT3041-1 Clontech Laboratories, Inc. Version No. PR6Z2173 3 SMART ™ PCR cDNA Synthesis Kit User Manual