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SMART™ PCR cDNA Synthesis KitUser Manual

Cat. No. 634902 PT3041-1 (PR752244)Published 15 May 2007

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 � Version No. PR75��44

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 35

XII. Related Products 36

Appendix: Virtual Northern Blots 37

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 technologyAllcommonlyusedcDNAsynthesismethodsrelyontheabilityofreversetranscriptase(RT)totranscribemRNAintosingle-stranded(ss)cDNAinthefirst-strand reaction.However,becauseRTcannotalways transcribe theentiremRNAsequence,the5'endsofgenestendtobeunder-representedincDNApopulations.ThisisoftenthecaseforlongmRNAs,especiallyifthefirst-strandsynthesisisprimedonlywitholigo(dT)primers,orifthemRNAhasapersistentsecondarystructure.IntheabsenceofRNAdegradation,truncatedcDNAmoleculespresentinlibrariesareoftenduetoRTpausingbeforetranscriptioniscomplete.Regardless,theSMARTmethodisabletopreferentiallyenrichforfull-lengthcDNAs.SMARTcDNAsynthesisstartswitheithertotalorpolyA+RNA.Amodifiedoligo(dT)primer (the3'SMARTCDSPrimer IIA)primes thefirst-strandsynthesisreaction(Figure1).WhenRTreachesthe5'endofthemRNA,theenzyme’sterminaltransferaseactivityaddsafewadditionalnucleotides,primarily deoxycytidine, to the 3' end of the cDNA.The SMART™ II AOligonucleotide,whichhasanoligo(G)sequenceatits3'end,base-pairswiththedeoxycytidinestretch,creatinganextendedtemplate.RTthenswitchestemplatesandcontinuesreplicatingtotheendoftheoligonucleotide(Chenchiket al., 1998).Theresultingfull-length,single-stranded(ss)cDNAcontainsthecomplete5'endofthemRNA,aswellassequencesthatarecomplementarytotheSMARTOligonucleotide.IncaseswhereRTpausesbeforetheendofthetemplate,theadditionofdeoxycytidinenucleotidesismuchlessefficientthanwithfull-lengthcDNA-RNAhybrids,thuspreventingbase-pairingwiththeSMART Oligonucleotide.The SMART anchor sequence and the poly Asequenceserveasuniversalprimingsitesforend-to-endcDNAamplification.Therefore,cDNAwithoutthesesequencesduetoprematurelyterminatedcDNAscausedbyincompleteRTactivity,contaminatinggenomicDNA,orcDNAtranscribedfrompolyA–RNA,willnotbeexponentiallyamplified.However, truncated RNAs that are present in poor quality RNA startingmaterialwill beamplified,whichwillcontaminatethefinalcDNAlibrary.

Synthesize SMART™ cDNA for a wide variety of applicationsThe first kit to feature SMART technology is the SMART cDNA LibraryConstructionKit (Cat.No.634901).This kit includes the components fordirectionalcloningoffull-lengthcDNA.Toexpandtherangeofapplications,the SMART PCR cDNA Synthesis Kit (Cat. No. 634902; Figure 2) wasintroduced shortly after.This kit allows you to synthesize high-qualitycDNAforlibraryconstructionusingyourownvectorandligationreagents.



I. Introduction continued

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

OtherapplicationsincludeClontechPCR-Select™cDNASubtraction(Cat.No.637401),“Virtual”Northernblots,andprobegeneration.PleasenotethattheSMARTII™AOligonucleotideisspeciallyengineeredforusewiththePCR-Selectmethod.cDNAgeneratedusingtheSMARTcDNALibraryConstructionKitcannotbeusedforPCR-SelectcDNAsubtraction.IntheSMARTlibraryconstructionprotocol,eachPCR-amplifiedcDNAmoleculehasanextraSMARTsequenceoneachendwhichdecreasestheefficiencyofsubtractionofamplifiedcDNA.

First-strand �synthesis by RT ��

Amplify cDNA by LD PCR�with PCR primer

Poly A+ RNA

polyA 3'

SMART II A�oligonucleotide

CDS primer

Double-stranded cDNA

Template switching �and extension by RT

polyA

polyAGGG

5'

GGG5'

GGG5'

CCC

dC tailing by RT

polyA

CCCGGG5'

Single�step

5'

5'

5'



I. Introduction continued

TheSMARTIIAOligonucleotideand3'SMARTCDSPrimerIIAprovidedintheSMARTPCRcDNASynthesisKiteachhaveanRsaIsitetofacilitateremovaloftheseidenticalsequencesfromthePCR-amplifiedcDNAmol-ecules.ThisUserManualincludestwoprotocolsforcDNAsynthesis.Thesepro-tocolshavebeendesignedtostrikeabalancebetweenmaintaininggenerepresentationandreducingnonspecificbackgroundamplification.Inthefirstprotocol(SectionsVandVI),undilutedfirst-strandsscDNAissubjectedtothefewestpossiblenumberofPCRcycles.ThisprotocolisidealforcDNAlibraryconstruction,wherehighrepresentationismostimportant(Zhuet al.,2001).Inthesecondprotocol(SectionsVIIandVIII),thefirst-strandsscDNAtemplateisdilutedandmorePCRcyclesareperformed.Thisgreatlyreduces nonspecific amplification, which is crucial for PCR-Select cDNAsubtractionandothernon-libraryapplications.Besuretochoosetheap-propriateprotocolforyourapplication.TheSMARTcDNAsynthesismethodisnowoptimizedforrapidamplifica-tionof cDNAends (RACE;Matzet al., 1999).TheSMART™RACEcDNAAmplificationKit(Cat.No.634914)integratesourMarathon®cDNAAmpli-ficationKit(Chenchiket al., 1995;1996)withourSMARTcDNAsynthesistechnologyandallowsyoutoperformboth5'and3'RACEusingeitherpolyA+ortotalRNA.ClontechhasrigorouslytestedournewSMARTRACEKittoverifythatitperformsevenbetterthantheMarathonKit(January1999Clontechniques).

Figure 2. Guide to SMART™ cDNA synthesis protocols.Besure to follow theappropriateprotocolforyourapplication.

Total or poly A+ RNA

SMART ds cDNA SMART ds cDNA

SMART cDNA synthesis(Sections VII & VIII)

SMART cDNA synthesis(Sections V & VI)

Otherapplications

PCR-SelectcDNA

subtractionVirtual

Northern blotscDNA libraryconstruction



I. Introduction continuedSMART™ cDNA synthesis for cDNA subtractionTheClontechPCR-SelectcDNASubtractionKit(Cat.No.637401)providesapowerfulmethodforidentifyingdifferentiallyexpressedgenes(Diatchenkoet al.,1996;Gurskayaet al.,1996).WhentotalRNAisusedforcDNAsynthesisbyconventionalmethods,ribosomalRNAistranscribedalongwiththepolyA+fraction,evenifsynthesisisoligo(dT)-primed.IfthiscDNAisusedwiththePCR-SelectKit,theexcessofribosomalRNAandlowconcentrationofcDNAcorrespondingtothepolyA+fractionresultsininefficientsubtrac-tivehybridization.However,cDNAgeneratedusingtheSMARTPCRcDNASynthesisKitcanbedirectlyusedforPCR-Selectsubtraction—eveniftotalRNAwasusedasstartingmaterial.

Virtual Northern blots and probesTheSMARTPCRcDNASynthesisKitmayalsobeusefulforresearcherswhowishtoanalyzetranscriptsizeandexpressionpatternsbyhybridizationbutlacksufficientpolyA+ortotalRNAforNorthernblots.Thisisespeciallyimportant for researchers who have isolated clones using the ClontechPCR-Select™KitandwhoalsoneedtoconfirmthedifferentialexpressionofcorrespondingmRNAs.“Virtual”NorthernblotscanbegeneratedusingSMARTcDNAinsteadoftotalorpolyA+RNA(Endegeet al.,1999),andcangiveinformationsimilartothatprovidedbystandardNorthernblots.FormoreinformationonVirtualNorthernblots,pleaseseetheAppendix.OtherapplicationsforSMARTcDNAincludepreparingprobesforhybrid-izationtohigh-densitycDNAorgenomicDNAarrays(Pietuet al.,1996)orfor thecDNAselection-basedpositional cloningmethod (Morganet al.,1992).Pleaseseethesereferencesformoreinformationabouttheseap-plications.

Advantage® 2 PCR Kit and PowerScript™ Reverse TranscriptaseWestronglyrecommendtheuseoftheAdvantage2PCRKits(Cat.Nos.639206&639207)forPCRamplification.ThesekitsincludetheAdvantage2PolymeraseMix,whichhasbeenspeciallyformulatedforefficient,accurate,andconvenientamplificationofcDNAtemplatesbylong-distancePCR(LDPCR;Barnes,1994).ThePolymeraseMixiscomprisedofTITANIUM™TaqDNA Polymerase—a nuclease-deficient N-terminal deletion of Taq DNApolymeraseplusTaqStart™Antibodytoprovideautomatichot-startPCR(Kellogget al.,1994)—andaminoramountofaproofreadingpolymerase.Thiscombinationallowsyoutoefficientlyamplifyfull-lengthcDNAswitha significantly lower error rate than that of conventional PCR (Barnes,1994).EachSMARTkitalsoincludesPowerScript™ReverseTranscriptase,apointmutantofMoloneymurineleukemiavirus(MMLV)reversetranscriptase(RT).PowerScriptRThassubstantiallyreducedRNaseHactivity,butretainswild-typepolymeraseactivity,soitcansynthesizelongercDNAfragmentsthanwild-typeMMLVRT.OurrigorouspurificationmethodalsoensuresthateachPowerScriptpreparationisnotcontaminatedwithRNaseandDNase.



II. List of Components

StoreCHROMASPINatroomtemperature.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

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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[pH

7.5],150mMNaCl,1mMEDTA). 3.Incubatethemixtureatroomtemperaturefor2–3hr. 4.Removeanddiscardthetoplayer. 5.Addanequalvolumeofchloroform:isoamylalcoholtotheremain-

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

at4°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•Microfiltration Columns (0.45μM)



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).

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

• Whateveryourapplicationmaybe,thesuccessofyourexperimentdependsonthequalityofyourstartingsampleoftotalorpolyA+RNA.ThereareseveralproceduresavailableforRNAisolation(Chomczynski&Sacchi,1987;Farrell,1993;Sambrooket al.,2001).Inaddition,ClontechoffersseveralkitsfortheisolationoftotalRNAandsubsequentisolationofpolyA+RNA.Alternatively,youmaywishtouseoneofourPremiumPoly A+ RNAs. For more information, visit our web site at www.clontech.com.

• Beforeyoubeginfirst-strandsynthesis,westronglyrecommendthatyoucheck the integrityof yourRNAbyelectrophoresinga sampleonaformaldehyde/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).

• WearglovesthroughouttheproceduretoprotectyourRNAandcDNAsamplesfromdegradationbynucleases.

• Thefirsttimeyouusethiskit,youshouldperformcDNAsynthesiswiththeControlHumanPlacentalTotalRNAprovidedinthekit,inparallelwith your experimental sample. Performing this control synthesisat leastoncewillverifythatallcomponents(especially thereversetranscriptase)areworkingproperlyandwillalsohelpyoutroubleshootanyproblemsthatmayarise.

IV. General Considerations



IV. General Considerations continued

• Thecyclingparametersinthisprotocolhavebeenoptimizedusinganauthorizedhot-lidthermalcycler.Optimalparametersmayvarywithdifferentthermalcyclersandtemplates.

• Toresuspendpelletsandmixreactions,gentlypipetthemupanddownandcentrifugethetubebrieflytodepositcontentsatthebottom.

• Vortexphenol:chloroformextractionstomix.

• Addenzymestoreactionmixtureslast,andthoroughlyincorporatetheenzymebygentlypipettingthereactionmixtureupanddown.

• DonotincreasetheamountofenzymeaddedorconcentrationofDNAinthereactions.Theamountsandconcentrationshavebeencarefullyoptimized.

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V. SMART™ cDNA Synthesis for Library Construction

Important: This protocol is designed for synthesizing SMART cDNA for library construction.Forotherapplications,includingClontechPCR-SelectcDNAsubtraction,consulttheprotocolinSectionsVIIandVIII.

Figure 3. Protocol guide for SMART™ cDNA synthesis for library construction. Ifagarose/EtBrgelanalysisofthedscDNAindicatesthatmorecyclesareneeded,simplyreturnthereac-tiontothethermalcyclerforafewmorecycles,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,combinethefollowingreagentsina

sterile0.5-mlreactiontube:

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

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

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

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

atthebottom.

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

7.Mix by gently pipetting and spin the tubes briefly in amicrocentrifuge.

8.Incubatethetubesat42°Cfor1hrinanairincubator.Note:Ifyouuseawaterbathorthermalcyclerforthisincubation,coverthereactionmixturewithonedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

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

a2-µlaliquotfromthefirst-strandsynthesistoaclean,prechilled,0.5-mlreactiontube.Placetubeonice.Ifyouusedmineraloilinyourfirst-strandreactiontube,becarefultotakethealiquotfromthebottom ofthetubetoavoidtheoil.

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




B. cDNA Amplification by LD PCR TableIprovidesguidelinesfortheoptimalnumberofthermalcyclesfor

agivenamountoftotalorpolyA+RNAusedinthefirst-strandsynthesis.TheseguidelinesweredevelopedusingtheControlHumanPlacentalTotalRNAandanauthorizedhot-lidthermalcycler;optimalparametersmayvarywithdifferenttemplatesandthermalcyclers.Usethefewest cyclespossible;overcyclingmayyieldnonspecificPCRproducts.Ifnecessary,undercyclingcanbeeasily rectifiedbyplacing thereactionback inthethermalcyclerforafewmorecycles(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.Aliquot98µloftheMasterMixintoeachreactiontubefromStep

A.10. 5.Mixcontentsbygentlyflickingthetubes.Centrifugetubesbriefly

inamicrocentrifuge. 6.Cap the tube, and place it in the preheated thermal cycler. If

necessary,overlaythereactionmixturewith2dropsofmineraloil.




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-mlmicrocentrifuge tube.StoretheremainderofthePCRmixtureat–20°C.Note: ProteinaseKtreatmentisnecessarytoinactivatetheDNApolymeraseactivitybeforeproceedingwiththeligationsteps.

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.AdddeionizedH2Otoresuspendthepellet.Theamountaddedwill

dependonyourcDNAlibraryconstructionprotocol.Note:Thispreparationofblunt-endedcDNAmaynowbeligatedtoanyadaptoryouchoose.ConsultyourprotocolforcDNAlibraryconstruction.



VI. Analysis of Results for Library Construction

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

1-kbDNAladdersizemarker(0.1µgrunonthesamegel).ForcDNAmadefromallmammalianRNAsources,theoverallsignalintensity(relativetothemarkerDNA)shouldberoughlysimilartothatshownforthecontrolexperimentinFigure4. If theintensityofthecDNAsmearismuchstrongerthanthatshownforthecontrol(relativeto0.1µgofsizemarker),especiallyifnobrightbandsaredistinguishable,thismayindicatethattoomanythermalcycleswereused—thatis,youhaveovercycledyourPCR(seeTroubleshootingGuide,SectionX.B).Ifthesmearismuchfainter(relativeto0.1µgofsizemarker)andthesizedistributiongenerallylessthan4kb,thentoofewthermalcycles(i.e., PCR undercycling) may be the problem (seeTroubleshootingGuide,SectionX.B).

2. Severalbrightbandscorrespondingtoabundanttranscripts ThepatternofbrightbandsshowninFigure4ischaracteristicofthe

dscDNAsynthesizedfromtheControlHumanPlacentalTotalRNAusingtheprotocoloutlinedinSectionV.AsindicatedbythearrowinFigure5,youshouldobserveastrong,distinctbandat900bp.AverystrongsmearofcDNAinthecontrolreactionwithoutthecharacteristicbrightbandsmaybeindicativeofPCRovercycling(seeTroubleshootingGuide,SectionX.B).Ifthecharacteristicbandsarepresentbutweak,thismaybeindicativeofPCRundercycling(seeTroubleshootingGuide,SectionX.B).ThenumberandpositionofthebandsyouobtainwithyourexperimentalRNAmaydifferfromthoseshownforthecontrolreaction.Furthermore,cDNApreparedfromsomemammaliantissuesources(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,suchasClontechPCR-Select™cDNASubtractionorVirtualNorthernBlots(SeeAppendix).TosynthesizeSMARTcDNAforlibraryconstruction,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.

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VII. SMART cDNA Synthesis Protocol continued

Important: IfyouareplanningtoproceedwiththeClontechPCR-SelectcDNASubtractionprotocol,werecommendreadingtheUserManualforcDNASubtractionbeforeproceedingwithfirststrandcDNAsynthesisusingtheSMARTmethod.ThecDNASubtractionKitsuppliesadifferentRNAcontrolthatshouldbeusedtosynthesizecDNAaccordingtothePCR-SelectUserManual(whichdescribesanon-SMARTmethod).Inaddition,usethecontrolprovidedinthiskittotroubleshootanyproblemsusingtheSMARTprotocol.Formoreinformationaboutusingthesecontrols,seeSectionVIII.DofthisUserManual.

A. First-Strand cDNA Synthesis 1.For each sample and the Control Human PlacentalTotal RNA,

combinethefollowingreagentsinasterile0.5-mlreactiontube:

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

5µl Totalvolume*For the control synthesis, add 1 µl (1 µg/µl) of Control Human PlacentalTotalRNA.

2.Mixcontentsandspinthetubebrieflyinamicrocentrifuge. 3.Incubatethetubeat70°Cinathermalcyclerfor2min. 4.Spinthetubebrieflyinamicrocentrifugetocollectcontentsatthe

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):

• Add 40 µl ofTE buffer if you used total RNA as startingmaterial.

• Add450µlofTEbufferifyouusedmorethan0.2µgofpolyA+RNAasstartingmaterial.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 �0 Version No. PR75��44



•Add90µlofTEbufferifyouusedlessthan0.2µgofpolyA+RNAasstartingmaterial.

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

B. cDNA Amplification by LD PCR TableIIprovidesguidelinesforoptimizingyourPCR,dependingon

theamountoftotalorpolyA+RNAusedinthefirst-strandsynthesis.TheseguidelinesweredeterminedusingtheControlHumanPlacentalTotalRNAandanauthorizedhot-lidthermalcycler;optimalparametersmayvarywithdifferenttemplatesandthermalcyclers.Todeterminethe optimal number of cycles for your sample and conditions, we

Figure 6. Optimizing PCR parameters for SMART™ cDNA synthesis. NotethatforsamplesnotusedforcDNAsubtraction,youwillonlyhavetwotubespersampleorcontrol:oneexperi-mentalsampletubeandone“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. PR75��44 �1



stronglyrecommendthatyouperformarangeofcycles:15,18,21,and24cycles(Figure6).

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

1.Preheatathermalcyclerto95°C. 2.For each reaction, aliquot the appropriate volume (seeTable II,

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. PR75��44


3.PrepareaMasterMixforallreactiontubes,plusoneadditionaltube.Combinethefollowingcomponentsintheordershown:

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

90µl Totalvolume

4.Mix well by vortexing and spin the tube briefly in amicrocentrifuge.

5.Aliquot90µlofthePCRMasterMixintoeachtubefromStep2. 6.Cap the tube, and place it in the preheated thermal cycler. If

necessary,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. Transfer15µlfromthe15-cyclePCRtoacleanmicrocentrifugetube(foragarose/EtBrgelanalysis).

b. Runthreeadditionalcycles(foratotalof18)withtheremaining85µlofthePCRmixture.

c. Transfer15µlfromthe18-cyclePCRtoacleanmicrocentrifugetube(foragarose/EtBrgelanalysis).

d. Runthreeadditionalcycles(foratotalof21)withtheremaining70µlofPCRmixture.

e. Transfer15µlfromthe21-cyclePCRtoacleanmicrocentrifugetube(foragarose/EtBrgelanalysis).

f. Runthreeadditionalcycles(foratotalof24)withtheremaining55µlofPCRmixture.




9.Electrophorese5µlofeachaliquotofeachPCRreactionalongside0.1µgof1-kbDNAsizemarkerona1.2%agarose/EtBrgelin1XTAEbuffer.Determinetheoptimalnumberofcyclesrequiredforeachexperimentalandcontrolsample(seeFigure7,SectionIX).

10.Retrievethe15-cyclePCRtubesfrom4°C,returnthemtothethermalcycler,andsubjectthemtoadditionalcycles,ifnecessary,untilyoureachtheoptimalnumber.

11.When the cycling is completed, analyze a 5-µl sample of eachPCRproductalongside0.1µgof1-kbDNAsizemarkerona1.2%agarose/EtBrgelin1XTAEbuffer.CompareyourresultstoFigure7toconfirmthatyourreactionsweresuccessful.

12.Add2µlof0.5MEDTAtoeachtubetoterminatethereaction. 13.Transfer7µlofyourrawPCRproducttoacleanmicrocentrifuge

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

YounowhaveSMARTdscDNAreadytouseforapplicationssuchasVirtualNorthernblottingorgenerationofcDNAprobes.ForPCR-SelectcDNAsubtraction,proceedwith the followingprotocol (StepVIII.A,below).

VIII. Protocol for Clontech PCR-Select™ cDNA Subtraction

A. Column Chromatography(PCR-SelectUsersonly!) 1.For every experimental sample and control, combine the two

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

2.Add an equal volume of phenol: choloroform:isoamyl alcohol(25:24:1).Vortexthoroughly.

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

4.Remove the top (aqueous) layer and place it in a clean 1.5-mltube.

5.Add700µlofn-butanolandvortexthemixturethoroughly.ButanolextractionallowsyoutoconcentrateyourPCRproducttoavolumeof40–70µl.Note:Additionoftoomuchn-butanolmayremoveallthewaterandprecipitatethenucleicacid.Ifthishappens,addwatertothetubeandvortexuntilanaqueousphasereappears.

6.Centrifugethesolutionatroomtemperatureat14,000rpmfor1min.

7.Removeanddiscardtheupper(n-butanolorganic)phase.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3041-1 �4 Version No. PR75��44


8.Ifyoudonotendupwithavolumeof40–70µl,repeatsteps5–7.Note:Ifyourvolumeis<40µl,addH2Ototheaqueousphasetoadjustvolumeto40–70µl.

9.InvertaCHROMASPIN1000Columnseveraltimestocompletelyresuspendthegelmatrix.Note:Checkforairbubblesinthecolumnmatrix.Ifbubblesarevisible,resuspendthematrixinthecolumnbufferbyinvertingthecolumnagain.

10.Removethetopcapfromthecolumn,andthenremovethebottomcap.

11.Placethecolumnintoa1.5-mlcentrifugetubeora17x100mmtube.

12.Discardanycolumnbufferthatimmediatelycollectsinthetubeandadd1.5mlof1XTNEbuffertothecolumn.

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.Transfer10µlofthisfractiontoacleanmicro-centrifugetubeandlabelthistube“SampleB”.Storeat–20°C.Usethisaliquotforagarose/EtBrgelanalysis(Step21,below).

20.Apply75µlof1XTNEbufferandcollecttheeluateinacleanmi-crocentrifugetube.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,which

arenecessaryforbothadaptorligationandsubtraction.

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



BeforeproceedingwithRsaIdigestion,setasideanother10µlofpuri-fieddscDNAforagarose/EtBrgelanalysistoestimatethesizerangeofthedscDNAproducts(Step4,below).Labelthistube“SampleD”.

1.AddthefollowingreagentstothepurifiedcDNAfractioncollectedfromtheCHROMASPINColumn(StepVIII.A.21):

10XRsaIrestrictionbuffer 36µlRsa I (10units) 1.5µl

2.Mixbyvortexingandspinbrieflyinamicrocentrifuge. 3.Incubateat37°Cfor3hr. 4.ToconfirmthatRsaIdigestionwassuccessful,electrophorese10

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

5.Add8µlof0.5MEDTAtoterminatethereaction. 6.Transfer10µlofthedigestedcDNAtoacleanmicrocentrifugetube,

labelthistube“SampleE”,andstoreat–20°C.YouwillcomparethissampletothePCRproductafterfinalpurification,asdescribedinSectionIX.D.

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

purificationsystem,suchasthoseofferedbyClontech(seeRelatedProducts,SectionXII).Alternatively,aphenol:chloroformextractionmaybeperformed;however,thismaydecreasetheefficiencyofthecDNAsubtraction.Thefollowingpurificationprocedurehasbeenop-timizedusingSMARTdscDNAandtheNucleoTrapPCRKit(Cat.No.636020;notincludedwithPCR-SelectKit).

Before you start:Add64mlof95%ethanoltotheBufferNT3forafinalconcentrationofapproximately85%.TheappropriatevolumeisalsolistedontheBufferNT3bottle.

1.AliquottheRsaI-digestedcDNA(SectionVIII.B.6,above)intotwoclean,1.5-ml microcentrifuge tubes (approximately 170 µl in eachtube).

2.VortextheNucleoTrapSuspensionthoroughlyuntilthebeadsarecompletelyresuspended.

3.Add680µlofBufferNT2and17µlofNucleoTrapSuspensiontoeachtubeofdigestionmixture.

4.Incubatethesampleatroomtemperaturefor10min.Mixgentlyevery2–3minduringtheincubationperiod.





5.Centrifugethesampleat10,000xgfor1minatroomtemperature.Discardthesupernatant.

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

7.Add680µlofBufferNT3tothepellet.Mixgentlytoresuspend.Centrifugethesampleat10,000xgfor1minatroomtemperature.Removethesupernatantcompletelyanddiscard.

8.RepeatStep7. 9.Centrifugethepelletagainat10,000xgfor1minatroomtem-

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

10.Add50µlofTEbuffer(pH8.0)tothepellet.Resuspendthepelletbymixinggently.Combinetheresuspendedpelletsintoonetube.Mixgently.

11.ElutetheDNAbyincubatingthesampleat50°Cfor5min.Gentlymixthesuspension2–3timesduringthisincubationstep.

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

13.Applythesupernatanttoamicrofiltrationcolumnthathasbeeninsertedintoa1.5-mltube.Centrifugefor5minanddiscardthecolumn.

14.Transfer6µlofthefilteredDNAsolutiontoaclean1.5-mlmicro-centrifugetubecontaining14µlofdeionizedH2O.Labelthistube“SampleF”andstoreat–20°C.YouwillusethissampletoanalyzetheSMARTcDNAafterpurification,asdescribedinSectionIX.D.

15.ToprecipitatetheDNA,add1/2volumeof4Mammoniumacetate(e.g., 50 µl for a 100-µl sample), then add 2.5 volumes of 95%ethanol(e.g.,375µlfor150µlsample+ammoniumacetate)totheremainingsamplefromStep14.

16.Vortexthemixthoroughlyandcentrifugethetubesat14,000rpmfor20minatroomtemperature.

17.Carefullyremoveanddiscardthesupernatant. 18.Overlaythepelletwith500µlof80%ethanol. 19.Centrifugethetubeat14,000rpmfor10min.Carefullyremovethe

supernatantanddiscard. 20.Airdrythepelletsfor5–10min.




21.Dissolvethepelletin6.7µlof1XTNEbuffer. 22.Transfer1.2µltoaclean1.5-mlmicrocentrifugetubecontaining

11µlofdeionizedH2O,labelthistube"SampleG,"andstoretheremainingsampleat–20°C.Use10µlofthedilutedDNAtoassesstheyieldofDNAbyUVspectrophotometry.Foreachreaction,weusuallyobtain1–3µgofSMARTcDNAafterpurification.Fortwotubes,youshouldobtainatotalof2–6µgofcDNA.Ifyouryieldislowerthanthis,performtheagarose/EtBrgelanalysisdescribedinSectionIX.D.

23.IfyourDNAconcentration is>300ng/µl,diluteyourcDNAtoafinalconcentrationof300ng/µlin1XTNEbuffer,andfollowtheadaptorligationstepinaccordancewiththeClontechPCR-SelectcDNAsubtractionprotocol.

24.YourdigesteddscDNAisnowreadyforadaptorligation,asdescribedinSection IV.Fof theUser Manual for the Clontech PCR-SelectcDNASubtractionKit(Cat.No.637401).BesuretoreadSectionVIII.DbelowforimportantcDNAsubtractioncontrolprocedures.

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

tractions.PleaserefertoSectionIVofthePCR-SelectUserManual.1. Controlsubtractionusingthehuman skeletal muscle poly A+ RNA

(includedinthePCR-SelectKit)Usetheconventionalmethod(asdescribedinthePCR-SelectUserManual) tosynthesizedscDNAfromthecontrolhumanskeletalmusclepolyA+RNAprovidedinthePCR-SelectKit.Then,setupa“mock”subtraction:useaportionofthehumanskeletalmusclecDNAasthedriver,andmixanotherportionwithasmallamountofthecontrolHae III-digestedφX174DNAfromthePCR-SelectKitasthetester.Thiscontrolsubtraction,whichisdescribedindetailinthePCR-SelectUserManual,isthebestwaytoconfirmthatthemultistepsubtractionprocedureworksinyourhands.

2.Controlsubtractionusingthe human placental total RNA (includedintheSMARTkit)

UsetheSMARTkit toamplify theControlHumanPlacentalTotalRNA; then,performamocksubtractionasdescribed forControlNo.1:useaportionofthehumanplacentalcDNAasthedriver,andmixanotherportionwithasmallamountofthecontrolHae III-di-gestedφX174DNAfromthePCR-SelectKitasthetester.IfControlNo.1works,butControlNo.2doesnot,youmayassumethattheSMARTcDNAamplificationand/orpurificationfailed.Inthiscase,tryreducingthenumberofPCRcyclesforthecDNAamplificationandtroubleshootyourpurificationprotocol(SectionVIII.C).



IX. Analysis for Clontech™ PCR-Select Subtraction

Figure7showsatypicalgelprofileofdscDNAsynthesizedusingtheControlHumanPlacentalTotalRNAandtheSMARTprotocoloutlinedinSectionVII.Asindicatedbythearrow,youshouldobserveastrong,distinctbandat900bp.Ingeneral,cDNAsynthesizedfrommammaliantotalRNAshouldappearona1.2%agarose/EtBrgelasamoderatelystrongsmearfrom0.5–6kbwithsomedistinctbands.Thenumberandpositionofthebandsyouob-tainwillbedifferentforeachparticulartotalRNAused.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,youmustoptimizethePCRcyclingparametersfor

yourexperiment,asdescribedinSectionVII.B(Figure6).ChoosingtheoptimalnumberofPCRcyclesensuresthatthedscDNAwillremainintheexponentialphaseofamplification.WhentheyieldofPCRproductsstopsincreasingwithmorecycles,thereactionhasreacheditspla-teau.OvercycledcDNAisaverypoortemplateforcDNAsubtraction.Undercycling,ontheotherhand,resultsinaloweryieldofyourPCRproduct.Theoptimalnumberofcyclesforyourexperimentisonecyclefewerthanisneededtoreachtheplateau.Beconservative:whenindoubt,itisbettertousefewercyclesthantoomany.

Figure 7. Analysis for optimizing PCR parameters. 5µlofeachPCRproductwaselectrophoresedona1.2%agarose/EtBrgelin1XTAEbufferfollowingtheindicatednumberofPCRcycles.Theoptimumnumberofcyclesdeterminedinthisexperimentwas17.LaneM:1-kbDNAladdersizemarkers,0.1µgloaded.Thearrowindicatesthestrongbandat900bptypicallyseenforhumanplacentaltotalRNA.

TotalRNAPolyA+RNA

M1518212415182124cycleskb

1232

1.6

1

0.5

←

Protocol No. PT3041-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR75��44 ��


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,and thermalcyclers.We strongly recommend that you optimize the number ofPCRcycleswithyourexperimentalsample(s)andtheControlHumanPlacentalTotalRNA.Trydifferentnumbersofcycles;then,analyzeyourresultsbyelectrophoresing5µlofeachproductona1.2%agarose/EtBrgelin1XTAEbuffer.

Figure7providesanexampleofhowyouranalysisshouldproceed.Inthisexperiment,thePCRreacheditsplateauafter18cycles;thatis,theyieldofPCRproductsstoppedincreasing.After21and24cycles,a smear appeared in the high molecular weight region of the gel,indicatingthatthereactionwasovercycled.Becausetheplateauwasreachedafter18cycles,theoptimalnumberofcyclesforthisexperi-mentwouldbe17.

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

oftheunpurifiedPCRproduct(SampleA,fromSectionVII.B.13)alongside10µlofthePCRproductpurifiedbycolumnchromatography(SampleB, fromSectionVIII.A.19)and10µlof thesecond fraction (SampleC, fromSectionVIII.A.20)ona1.2%agarose/EtBrgel.Compare theintensitiesofSampleAandSampleB,andestimatethepercentageofPCRproductthatremainsaftercolumnchromatography.TheyieldofcDNAaftercolumnchromatographyistypically50percent.Ifyouryieldis<30percent,checktoseeifitispresentinthesecondfraction,SampleC.IfthissecondfractionhasahigheryieldofcDNAthanthefirst,combinethefractionsandproceedwithSectionVIII.B.OtherwiseifthecDNAisnotpresentinSampleC,repeatthePCRandcolumnchromatographysteps.

C. Rsa I Digestion (Section VIII.B) ToconfirmthatRsaIdigestionwassuccessful,electrophorese10µl

ofuncutdscDNA(SampleD,fromSectionVIII.B)alongside10µlofRsa I-digestedcDNA (fromSectionVIII.B.4)ona1.2%agarose/EtBrgel.Compare theprofilesofboth samples.Before Rsa I digestion,dscDNAshouldappearasasmearfrom0.5–10kbwithbrightbandscorresponding toabundantmRNAs. (For someRNAsamples fromnonmammalianspecies,thesizedistributionmaybeonly0.5–3kb.)AfterRsaIdigestion,thesmearshouldrangefrom0.1–2kb.ThisresultwillbesimilartothatshownintheUserManualforthePCR-SelectKit.



D. Purification of Digested cDNA (Section VIII.C) ToanalyzetheyieldofpurifiedSMARTcDNA,electrophorese10µlof

RsaI-digestedcDNAbeforepurification(SampleE,fromStepVIII.B.6)alongside10µlofpurifieddilutedcDNAbeforeethanolprecipitation(SampleF,StepVIII.C.14)and1.8µlofpurifieddilutedcDNAafterethanolprecipitation(SampleG,fromStepVIII.C.22)ona1.5%agarose/EtBrgel.ComparetheintensitiesofthesamplesandestimatewhatpercentageofRsaI-digestedPCRproductremainsafterpurificationandethanolprecipitation.TheyieldofcDNAafterpurificationusingtheNucleoTrapPCRKitandethanolprecipitationistypically70percent.Ifyouryieldis<30percent,troubleshootyourpurificationprotocolorconsultthetroubleshootingguideoftheUserManualforthatparticularpurifica-tionkit.

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

b. Youmayhavemadeanerrorduringtheprocedure,suchasusing a suboptimal incubation temperature or omitting anessentialcomponent.Carefullychecktheprotocolandrepeatthefirst-strandsynthesisandPCR.

c. The conditions and parameters for PCR may have beensuboptimal.TheoptimalnumberofPCRcyclesmayvarywithdifferentPCRmachines,polymerasemixes,orRNAsamples.If your PCR reaches its plateau after 24 cycles or more, theconditionsofyourPCRmaynotbeoptimal.ChecktheprotocolandrepeatthePCRusingafresh2-µlaliquotofthefirst-strandproduct.

2.Poor yield or truncated PCR product from your experimentalRNA

If thereactionwiththeControlHumanPlacentalTotalRNAwassuccessful,butyourexperiment failed,yourexperimentalRNAsamplemaybetoodiluteordegraded,ormaycontainimpuritiesthatinhibitfirst-strandsynthesis.IfyourRNAsamplewaspreparedfrom a nonmammalian species, the apparently truncated PCRproductmayactuallyhave thenormal sizedistribution for thatspecies.Forexample,forinsects,thenormalRNAsizedistributionmaybe<2–3kb.Ifyouhavenotalreadydoneso,electrophoresea sample of your RNA on a formaldehyde/agarose/EtBr gel todetermineitsconcentrationandanalyzeitsquality(seeSectionIVformoredetails).

a. TheconcentrationofyourexperimentalRNAis low,but thequalityisgood.

Repeat the experiment using more RNA and/or more PCRcycles.

b. Your experimental RNA has been partially degraded(by contaminating RNases) before or during first-strandsynthesis.

X. Troubleshooting Guide



RepeattheexperimentusingafreshlotorpreparationofRNA.CheckthestabilityofyourRNAbyincubatingasmallsamplefor2hrat42°C.Then,electrophoreseitonaformaldehyde/aga-rose/EtBrgelalongsideanunincubatedsample.IftheRNAisdegradedduring incubation, itwillnotyieldgoodresults inthefirst-strandsynthesis.Inthiscase,re-isolatetheRNAus-ingadifferenttechnique,suchasoneemployedbyourRNAisolationkits(seeRelatedProductsfororderinginformation).Severaladditionalroundsofphenol:chloroformextractionmaydramaticallyincreaseRNAstability.

c. YourexperimentalRNAsamplecontainsimpuritiesthatinhibitcDNAsynthesis.

Insomecases,ethanolprecipitationofyourexistingtotalRNA,followedbywashingtwicein80%EtOH,mayremoveimpuri-ties.Ifthisfails,reisolatetheRNAusingadifferenttechnique,suchasoneemployedbyourRNAisolationkits(seeRelatedProductsfororderinginformation).

B. Special Considerations for Library Construction (Sections V & VI) 1.LowyieldofPCRproduct a. ToofewthermalcycleswereusedinthePCRstep.Anotherin-

dicationofPCRundercyclingisacDNAsizedistribution<3kbifthemRNAsourcewasmammalian.(Forsomesources,suchasmanyinsectspecies,thenormalmRNAsizedistributionmaybe<2–3kb.)Ifyoususpectthatundercyclingistheproblem,in-cubatethePCRmixturefortwomorecyclesandrechecktheproduct. If you already used the maximum recommendednumberofcyclesindicatedinTableI,increasebythreemorecycles.IfincreasingthenumberofcyclesdoesnotimprovetheyieldofPCRproduct,repeatthePCRusingafresh2-µlaliquotofthefirst-strandproduct.

b. IfyoustillobtainalowyieldofPCRproduct,itmaybeduetoa lowyieldoffirst-strandcDNA.Possibleproblemswiththefirst-strandreactionincludeamistakeintheprocedure(suchasusingasuboptimal incubation temperatureoromittingacomponent)ornotusingenoughRNAinthereaction.Itisalsopossible that the RNA has been partially degraded (by con-taminatingRNases)beforeorduringthefirst-strandsynthesis.Reminder:problemswiththefirst-strandcDNAsynthesiscanbemoreeasilydiagnosedifyouperformparallelreactionsus-ingtheControlRNAprovidedinthekit.IfgoodresultswereobtainedwiththeControlRNAbutnotwithyourexperimentalRNA,thentheremaybeaproblemwithyourRNA.

Theeasiestwaytocheckthequalityofthefirst-strandcDNAisbyusingasmallsampleofitasaPCRtemplatewith3'and5'

X. Troubleshooting Guide continued



gene-specificprimers,suchasHumanβ-ActinControlAmplimers(Cat.No.639001,Cat.No.639002).Ifthefirst-strandsynthesishasbeensuccessful,aPCRproductoftheexpectedsizewillbegenerated.

2.NobrightbandsdistinguishableinthePCRproduct FormostmammalianRNAsources,thereshouldbeseveralbright

bandsdistinguishableagainstthebackgroundsmearwhenasampleofthePCRproductisrunonagel.Ifbrightbandsareexpectedbutarenotvisible,andthebackgroundsmearisveryintense,youmayhaveovercycledyourPCR.Ifyoususpectthatyourproblemis due to overcycling, then the PCR step (SectionV.B) must berepeatedwithafresh2-µlsampleoffirst-strandcDNA,using2–3fewercycles.

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

For troubleshooting the actual PCR-Select subtraction procedure,pleaserefertotheUserManualfortheClontechPCR-Select™cDNASubtractionKit.Here,weprovideatroubleshootingguideforprepar-ingSMARTcDNAforsubtraction(describedinSectionVIIandVIII).

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

CHROMASPINcolumn,orcollectedthewrongvolumeofbuf-ferfromthecolumn.Carefullychecktheprotocolandrepeatcolumnchromatography.

b. Yourcolumnmayhaveleakedduringshipping.Ifyourcolumncontainslessthan750µlofmatrix,discarditanduseanothercolumn.

2.FailureofRsaIdigestion(SectionVIII.B) Ifthesizedistributionofyoursampleand/orcontrolcDNAisnot

reducedafterRsaIdigestion,checktherecipeforTNEbuffer.IfyouusedthecorrectrecipeforTNEbuffer,performphenol:chloroformextractionandethanolprecipitation;then,repeattheRsaIdiges-tion.

3.LowyieldofcDNAafterpurificationofdigestedcDNA(SectionVIII.C)

Possiblereasonsforlowyieldincludethefollowing: a. LossofcDNAduringpurification.Troubleshootyourpurification

procedure. b. LossofcDNAduringethanolprecipitation.Checkthevolumes

oftheammoniumacetateandethanol.Repeatpurificationandethanolprecipitation.




c. YourPCRdidnotreachtheplateau(i.e.,thereactionwasun-dercycled).PerformmorePCRcycles.OptimizethenumberofcyclesasdescribedinSectionIX.




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-stepmethodofRNAisolationbyacidguanidiniumthiocyanate-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)Suppressionsubtractivehybridization: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)RepresentativecDNAlibrariesandtheirutility ingeneexpressionprofiling.BioTechniques 26:542–550.

Farrell,Jr.,R.E.(1993)RNA Methodologies—A Lab Guide for Isolation and Characterization(AcademicPress,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:CloningofJurkatcell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Anal. Biochem.240:90–97.

Kellogg, D. E., Rybalkin, I., Chen, S., Mukhamedova, N., Vlasik, T., Siebert, P.&Chenchik,A.(1994)TaqStartAntibody:HotstartPCRfacilitatedbyaneutralizingmonoclonalantibodydirectedagainstTaqDNApolymerase.BioTechniques16:1134–1137.

Lukyanov,S.A.,Gurskaya,N.G.,Tarabykin,V.S.&Sverdlov,E.D.(1994)Highlyefficientsub-tractivehybridizationofcDNA.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.,Houlgatte,R.,Soularue,P.&Auffray,C.(1996)NovelgenetranscriptspreferentiallyexpressedinhumanmusclesrevealedbyquantitativehybridizationofahighdensitycDNAarray.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)Reverse transcriptasetemplateswitching:ASMARTTMapproachforfull-lengthcDNAlibraryconstruction.BioTech-niques30:892–897.



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



AftercloningyoursubtractedcDNAfragments,youshouldconfirmthattheyrepresentdifferentiallyexpressedgenes.Typically,thisisaccomplishedbyhybridizationtoNorthernblotsofthesameRNAsamplesusedasdriverandtesterforsubtraction.If,however,youhavelimitedsamplematerial,youmaywishtouseVirtualNorthernblotsforanalysis.ByusingthesameSMARTPCR-amplifiedtesteranddrivercDNAusedforsubtraction,youcanobtaininformationthatissimilartothatprovidedbystandardNorthernanalysis.EvenifacDNAdoesnotgiveasinglebandwhenhybridizedtoaVirtualNorthernblot,youcanstilldetectwhetherornotitisdifferentiallyexpressed.MultiplebandsonaVirtualNorthernblotmayresultfromdifferentcauses.ThecDNAmaybelongtoamulti-genefamily,ormaycontainanucleotiderepeat. Alternatively, a truncated copy of the gene may be present.Todistinguishbetweenthesepossibilities,analysisshouldalsoincludeothermethods,suchasgenomicDNAsequencingorRACE.

To prepare a Virtual Northern blot, electrophorese your SMART PCR-amplified cDNA (before purification) on an agarose/EtBr gel and use aSoutherntransferontoanylonmembrane(seeSambrooket al.,1989).AtClontech,weusetheTurboBlotterequipmentandprotocolfromSchleicher&Schuell.Figure8showshowVirtualNorthernblotscanbeusedtoconfirm

Appendix: Virtual Northern Blots

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

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Notice to Purchaser

Clontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,in vitrodiagnosticpurposes,therapeutics,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,modification,reformulation,orresaleofthereagentsprovidedinSMART™Productsisnotpermitted.ForinformationonlicensingSMART™Technol-ogy forcommercialpurposes,pleasecontacta licensing representativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.TurboBlotter™isatrademarkoftheWhatmanGroup.NucleoTrap®andNucleoSpin®areregisteredtrademarksofMACHEREY-NAGELGmbH&Co.KG.Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.,unlessnotedotherwise.ClontechisaTakaraBioCompany.©2007

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