Creator™ DNA Cloning Kits User Manual - College of …web.as.uky.edu/biology/faculty/mirabito/BIO 510 Fall 2007...human and other mammalian sources. Using a full-length sequencing

Creator™ DNA Cloning KitsUser Manual

PT3460-1 (PR631583)Published 23 March 2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 � Version No. PR631583

Creator™ DNA Cloning Kits User Manual

Table of Contents

I. Introduction 4

II. List of Components 12

III. Additional Materials Required 13

IV. Creator™ DNA Cloning System 14

A. Beforeyoustart 14

B. CreatorDNACloningProcedure 14

C. ColonypCR 15

V. Typical Results 17

VI. Troubleshooting Guide 18

VII. References 21

VIII. Related Products 22

Appendix A: Creator™ Donor & Control Vector Maps 24

Appendix B: Creator™ Donor Vector MCSs 27

Appendix C: Competent Cells 29

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List of Figures

Figure1. LoxPsequence. 4

Figure2. FlowchartoftheCreatorDNACloningSystem. 5

Figure3. FlowchartoftaggingusingpDNR-Dual. 8

Figure4. TypicalPlatingResult 16

Figure5. TheCreatorsystemeasilygenerates10differentconstructs inoneday. 17

Figure6. Amplificationacrossarecombinationjuncture. 18

Figure7. Typicaltestresultsforsuccessfulrecombination. 20

Figure8. MapofpDNR-1rDonorVector. 24

Figure9. MapofpDNR-1r-LucControlVector. 24

Figure10.MapofpDNR-DualDonorVector. 25

Figure11.MapofpDNR-Dual-LucControlVector. 25

Figure12.MapofpDNR-CMVDonorVector. 26

Figure13.MapofpDNR-CMV-LacZControlVector. 26

Figure14.MCSofpDNR-1rDonorVector. 27

Figure15.MCSofpDNR-DualDonorVector. 27

Figure16.MCSofpDNR-CMVDonorVector. 28

List of Tables

TableI. CreatorAcceptorVectors 9

TableII. CreatorDonorVectors 10

TableIII. RecommendedCompetentCells 29

Table of Contents continued

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I. Introduction

TheCreator™ DNA Cloning Kits providearevolutionarysystemfortransfer-ringa targetgenedirectly intomultipleexpressionvectors,without theneedfortime-consumingsubcloningsteps.WiththeCreatorDNACloningKits,youcanstudynovelprotein interactions, tetracycline-regulatedexpression,signaltransduction,retroviralexpression,fluorescentproteintagging,andmanyotherapplications,simultaneously.

Cre-lox site-specific recombinationTheCreatorSystemusesCre-loxPsite-specificrecombinationtocatalyzethetransferofatargetgenefromaDonorVectorplasmidcontainingyourgeneofinterestÑtoanAcceptorVector,aplasmidcontainingregulatoryelementsofthedesiredhostexpressionsystem(Figure2).Cre,a38-kDarecombinaseproteinfrombacteriophageP1,mediatesrecombinationbetweenorwithinDNAsequencesatspecificlocationscalledloxP sites(Sauer,1994;Abremskiet al.,1984).Thesesitesconsistoftwo13-bpinvertedrepeatsseparatedbyan8-bpspacerregionthatprovidesdirectionalitytotherecombinationreaction(Figure1).The8-bpspacerregioninthe loxPsitehasadefinedorientationwhichforcesyourgenetobetransferredinafixedorientationandreadingframe.

DonorVectorscontaintwo loxPsites,whichflankthe5'endoftheMCSandthe5'endoftheopenreadingframeforthechloramphenicolresistancegene(Cmr;Figure2).DonorVectorsalsocontaintheampicillingeneforpropagationandselectioninE. coli,andthesucrasegenefromB. subtilis (SacB)forselectionofcorrectrecombinants.AcceptorVectorscontainasingle loxPsite,followedbyabacterialpromoter,whichdrivesexpressionofthechloramphenicolmarkerafterCre-lox-mediatedrecombination.Thegeneofinterest,oncetransferred,willbecomelinkedtothespecificexpressionelementsforwhichtheAcceptorVec-torwasdesigned.Furthermore,ifthecodingsequenceforthegeneofinterestisinframewiththeupstreamloxPsiteintheDonorVector,itwillautomaticallybeinframewithallpeptidesintheAcceptorVector.Therefore,youonlyneedtodeterminethecorrectreadingframeonce,andyourtargetgenewillalwaysbetransferredinthecorrectreadingframeandorientationintheAcceptorVectorafterrecombinationandselection.

Figure 1. LoxP sequence, showing reading frame.

ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA T

8-bp spacer region

Left inverted repeat Right inverted repeat



I. Introduction continued

Figure 2. Flow chart of the Creator™ DNA Cloning System showing a representative Donor Vector, pDNR-1r.

Acceptor vectorloxP

Prok.Promoter

Promoter/Peptide

Tag

Kanr

pDNR-1r Gene ofinterest

pDNR-1r

loxP

loxP

MCS

Insert gene of interest

SacBAmpr

pUC ori

Desired functionalexpression vector

Retroviralexpression

Cmr

loxP

loxPSacB

Kanr

loxP Prok.Promoter

Promoter/Peptide

Tag

pUC ori

Gene ofinterest

loxP

Cmr

Ampr

Cmr

Acceptor vectors available for:• Matchmaker two-hybrid analysis• High-level constitutive expression• Living Colors™ fluorescent protein tagging• Tet-regulated inducible expression• Retroviral expression• Bacterial expression

Cre-lox recombination•

Cm/Sucrose selection

Donor Vector




Creator™ DNA cloning & expression systemFigure2providesanoverviewoftheCreatorDNACloning&ExpressionSystem.TotransferyourgenefromtheDonorVectorintoanyAcceptorVector,simplyincubatetheDonorVectorcontainingyourgeneofinterestandanAcceptorVectorwithCreRecombinase(seeTablesI&IIforalistofCreatorAcceptorandDonorVectors).CrebindstotheloxPsitesonboththeDonorVectorandAcceptorVector,cleavestheDNA,andcovalentlyattachesitselftotheDNA.ThenCrecatalyzesstrandexchangeandligationoftheDNAsothatthegeneistransferredfromtheDonorVectorintotheAcceptorVector.Asaresult,arecombinantconstructiscreatedthatexpressesyourgeneofinterestinthedesiredhostsystem.Chlor-amphenicolandsucroseselectionletsyouharvestdesiredrecombinantcoloniesthatcontainadirectionallycorrectgeneinsert.ClonescontainingtheremainingDonorVector,withoutyourgeneinsert,willexpressSacB,andtherefore,cannotbegrownonmediacontainingsucrose.

Transfer target genes easilyTraditionalcloningpracticesrequireseveraldaysoftediousrestrictionenzymedigestion,fragmentpurification,andre-ligationprocedures.TheCreatorSystemissosimplethatinjustonedayyoucancreatemultipleconstructsthatarereadyforimmediateuseinfunctionalstudies.

Inseparatetubes,combineappropriateAcceptorVectorswithaDonorVector,containing your gene of interest, and Cre Recombinase. Incubate tube(s) atroomtemperaturefor15minutes.Next,theenzymeisheat-killedfor5minutesat70°C.TransformcompetentcellswithanaliquotoftheCreatorreactionmixture.After30–60minutesinSOCorLBmedium,platecellsonagarplatescontainingchloramphenicolandsucroseforselectionofdesiredrecombinants.Aftercoloniesareselected,youcanimmediatelyprepareDNAforfurtherstudies.Ifdesired,recombinantplasmidscanbefurtherpropagatedineitherchloramphenicolortheantibioticthatisappropriatefortheresistancemarkercarriedbytheAccep-torVector.

Creator gives you access to multiple expression systemsWithCreator you canplace your genewithinmultiple systems for functionalanalysis.ClontechoffersAcceptorVectorsformanyofourmostpowerfulsys-tems (Table I). The Matchmaker™Acceptor Vectors enable you to discovernovelprotein-proteininteractionsusingtheMatchmakerTwo-HybridSystem3.TheLivingColors™AcceptorVectorsallowyoutogenerateC-terminalfusionsofyourtargetgenetoafluorescenttagforproteinlocalizationstudies.Fordose-dependentinducibleexpressionstudiesofyourprotein,transferyourgeneintoanAcceptorVectorthatiscompatiblewithbothTetandtheretroviralRevTet™GeneExpressionSystems.TheIRESbicistronicexpressionAcceptorVectorshaveanIRES(internalribosomalentrysite)sequenceandaconstitutiveCMVpromotertoproduceabicistronicmessageforhighexpressionofyourprotein

Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 �



inmammaliancells.ThepLP-PROTet-6xHNAcceptorVector(Cat.No.631201)yieldshigh inducibleprotein levels inbacterialcells foreasypurificationwithTALON®resin.FormoreinformationontheseandotherCreatorvectors,pleaserefertoTablesIandIIorvisittheClontechpageat www.clontech.com.

CompleteSystemscompatiblewithCreatorareavailableforseveralofourgeneexpressionsystems.Thesecompletekitsofferastreamlinedapproachtoexpress-ingyourtargetgene.SeeSectionVIII,RelatedProductsforacurrentlist.

Creator Cloning Kits TheCreator™pDNRCloningKit(Cat.No.631615)isareformulatedversionofouroriginalCreatorCloningKit.TheDonorVectorinthiskit,pDNR-1risdesignedtoincreaserecoveryofrecombinantvectorscontaininginsert.ThepDNRCloningKitcanbeusedtotransferatargetgeneintoanyofourwiderangeofexpressionAcceptorVectorinthepresenceofCrerecombinase.

Donor Reporter Vectors generate reporter constructs in 15 minutesOurDonorReporterVectors,pDNR-SEAP,andpDNR-LacZallowyoutorapidlygeneratereporterconstructsforanygeneexpressionsystem.Usingthestandard15minuterecombinationreaction,CrerecombinasemediatesthetransferofthereporterfromtheDonorVectortoanyexpressionAcceptorVector.TheresultingreporterAcceptorVectorsaresuitableformanyuses,includingtransfectionef-ficiencycontrolsorquantifiersofinductionofgeneexpression.Thesereportersarealsoidealfornormalizingyourdata.

Ready-made Creator™ LibrariesÑor generate your ownTheCreator™SMART™cDNALibrariesweredevelopedtoserveasthefoundationfortheMammalianGeneCollection(MGC)project,ajointeffortoftheNationalInstitutesofHealth(NIH)andtheNationalCancerInstitute(NCI).Thisprojectaimstofacilitatethesearchforandisolationofyourtargetgenebyprovidingresearcherswithafullsetofinexpensive,full-lengthclonesandsequencesfromhumanandothermammaliansources.Usingafull-lengthsequencingpipeline,theprojectproduces5,000highlyaccuratefull-lengthsequencesperyear.Thesesequencesareanalyzedbyabioinformaticsgroup,andthenmadeavailabletotheresearchcommunitythroughGenBankandtheMGCwebsite(http://mgc.nci.nih.gov).Byprovidingthebiomedicalresearchcommunitywithinexpensive,full-lengthclonesinarapid-cloningformat,theMGCprojecthopestoacceler-atethefunctionalanalysisofgenesidentifiedbymammaliangenomeprojects.Currently,nearly1,000 full-length,sequenced-verifiedcDNAs thatareclonedintothepDNR-LIBVectorfromtheCreatorSMARTcDNALibrariesareavailablethroughtheI.M.A.G.E.Consortium.Thelibrariescanbequicklyscreened,andyourfull-lengthcDNAclonesisolatedbystandardprocedures.Thetargetgeneisthenefficientlytransferredintoavarietyofexpressionvectorsforfunctionalanalysis.TheCreatorLibrariesareavailablefromnumerouscancerandnormaltissuesfromvarioussources.




Figure 3. Flowchart of tagging using pDNR-Dual. ThepDNR-DualDonorVectorcontainsasplicedonor(SD)sitedirectlydownstreamoftheMultipleCloningSite(MCS).TheSDsite,whichistrans-ferredfrompDNR-Dualalongwiththegeneofinterest,mediatesthefusionofthegenetothetagintheAcceptorVectorthroughintronsplicing.Asaresult,atranscriptiscreatedthatexpressesthetagasa3'fusiontoyourgeneofinterestinaeukaryotichostsystem.

DesiredAcceptor VectorloxP

Prok.Promoter

5' Tag

pDNR-DualDonor Vector

Gene ofinterest

Expresssion Clone

loxP

loxPSacB

Cmr

loxPProk.

Promoter

5' Tag

Gene ofinterest

loxP

Cmr

3' Tag

SASD

3' Tag

SA

SD

Start Stop

Gene of interest

5' Tag 3' Tag

Promoter

Promoter

Start Stop

Gene of interest

5' Tag 3' Tag

SASD

Cmr loxP loxP Prok.

Promoter

mRNA (before splicing)

mRNA (after splicing)

loxP

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TABLe I. CReAToR™ ACCePToR VeCToRS

Acceptor Vector Promoter/Features Functional Application

pLP-GADT7 ADH1/GAL4activationdomain ExpressfusionstoGAL4ADtostudy proteininteractionsthroughtwo-hybrid screening.

pLP-GBKT7 ADH1/GAL4DNA-bindingdomain ExpressfusionstoGAL4DNA-binding domaintostudyproteininteractions throughtwo-hybridscreening.

pLP-AcGFP1-C CMV/fusionsofgeneto ExpressfusionstoAcGFP(Aequora C-terminalofAcGFP coerulescens greenfluorescentprotein) tostudylocalizationoftheproteinof interestinlivecells;nodyesorcofac- torsrequired.

pLP-IReSneo CMV/IRES,neoselectionmarker Constitutivemammalianexpression withsingletranscriptforbothgeneof interestandneoselectionmarker.

pLP-TRe2 Inducibletet-responsive High-level,regulatedpromoter expressionvector forexpressioninmammaliancells.

pLP-RevTRe Inducibletet-responsivepromoter High-level,regulatedretroviral inretroviralexpressionvector expressioninmammaliancells. withhygselection

pLP-LNCX CMVpromoterinretroviral Constitutiveretroviralexpression. expressionvectorwithneoselection

pLP-CMVneo CMVpromoterwithneoselection Constitutivemammalianexpression

pLP-CMV-Myc CMV/C-terminalfusionstoMyc ExpressfusionstoMyctagfordetection

pLP-CMV-HA CMV/C-terminalfusionstoHA ExpressfusionstoHAtagfordetection

pLP-PRoTet Inducibletet-responsive High-level,regulatedpromoterfor -6xHN expressionvector expressioninbacteria throughtwo-hybridscreening.

pLP-BacPAK9 Prokaryoticpromoter Constitutivebaculoviralexpression baculoviralconstruct

*OnlycompatiblewithgenesclonedinpDNR-DualDonorVector.



TheCreator™SMART™cDNALibraryConstructionKit(Cat.No.634903) pro-videsadependablemethodforproducinghigh-quality,cDNAlibrariescompat-iblewithCreatorSystemsspecificforyourresearchneeds.SMARTtechnologymakesitpossibletogeneratefull-length,directionallyclonedcDNALibrariesfromnanogramsoftotalorpolyA+RNA.UsingtheCreatorCloningSystem,isolatedclonesfromfinishedlibrariescanbetransferreddirectlytoexpressionAcceptorVectorsforfunctionalanalysisÑwithouttheneedforsubcloning.

Advantages over other Cre-lox systemsIntheCreatorSystem,two loxPsitesintheDonorVectorflanktheMCSandchloramphenicolopenreadingframe,soonlythissmallregionoftheDonorVec-toristransferredtotheAcceptorVector.Inothersystems,thereisonlyoneloxP siteintheDonorVectorcausingboththedonorandAcceptorVectorstofuseintoonelargeplasmid.Thus,singleloxPsite-basedDonorVectorsarenotcompat-iblewithretroviralandIRESAcceptorVectorsduetosizeconstraints.ThesmallexpressionvectorproducedbytheCreatorSystemiseasiertouseandisidealforavarietyofdownstreamapplications,includingretroviralexpression.

CreatordoesnotrequirePCRcloningmethodssothereisnoneedtosequencetheentiregene insert.Yoursequenceremains intact,anespecially important


TABLe II. CReAToRª DoNoR VeCToRS

Donor Vector Promoter/Features Functional Application

pDNR-1r T7promoter,M13F/Rprimersites, T7RNApolymeraseprimer/promoter SacBselection siteupstreamofMCSforin vitrotran- scription/translationofgeneofinterest

pDNR-Dual T7promoter,M13forwardsite, T7RNApolymeraseprimer/promoter SDsite,6xHNc-tertag, siteupstreamofMCSforin vitrotran- SacBselection scription/translation of gene of interest C-ter tagging by intron splicing in Eukaryotesandhasbuiltin6xHNtagat C-terforbacteria.

pDNR-CMV CMVpromoter,M13F/Rprimersites, T7RNApolymeraseprimer/promoter T7promoter,SacBselection siteupstreamofMCSforin vitrotran- scription/translation of gene of interest, CMVpromoter forexpression testing in mammaliancellspriortotransfer

pDNR-LIB T7promoter,M13F/Rprimersites, smallerpDNR1vector,T7RNApoly- SacBselection merase primer/promoter site upstream of MCS for in vitro transcription/transla tion of gene of interest, designed for library construction using SMART™ LibraryConstructionKit

pDNR- LacZ SacBselection DonorVectorwithReportergenesforpDNR- SeAP enzymaticquantitationorcellstaining assay



featureforgenesundergoingfunctionalstudies.OthersystemsrequireyoutosequencethefullinsertbecausetheirproceduresusePCR-basedcloning,whichmayintroduceerrorsduetothelowfidelityofsomethermostablepolymerases.AnotheradvantageoftheCreatorSystemisthatyoucantransformanystan-dardcompetentcelllines,suchasDH5αcells,afterperformingCre-mediatedrecombination.Inaddition,CreRecombinaseistheonlyenzymenecessaryfortherecombinationreaction,anditdoesnotrequireadditionalcofactors,nordoesithaveapreferenceforlinearoversupercoiledDNA.

TheCreatorDNACloningKits includeCreRecombinase, aDonorVector, aControlDonorVector,10XBSA,and10XCreReactionBuffer.Eachkitincludessufficientreagentstoperform10reactions.Inaddition,CreRecombinasecanalso be purchased separately (Cat. No. 631614; sufficient for 20 reactions).ClontechoffersavarietyofcompetentcellsandawiderangeofAcceptorVec-tors.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 1� Version No. PR631583


Storevectorsat–20°C.Storeallothercomponentsat–70°C.

Afterthefirstuse,storeCreRecombinaseat–20°Cinanon-frost-freefreezer.

Creator DNA Cloning Kits each include sufficient reagents to perform10reactionsasdescribedinthisUserManual.

Creator pDNR Cloning Kit(Cat.No.631615)

• 20 µg pDNR-1rVector(500ng/µl)• 20 µg pDNR-1r-LucControlVector(500ng/µl)• 10 µl CreRecombinase• 100 µl 10XCreReactionBuffer• 100 µl 10XBSA(1mg/ml)• pDNR-1rVectorInformationPacket(PT3616-5)

Cre Recombinase(Cat.No.631614)• 20 µl CreRecombinase• 100 µl 10XCreReactionBuffer• 100 µl 10XBSA(1mg/ml)

II. List of Components



Thefollowingmaterialsarerequiredbutnotsupplied:

• Acceptor Vectors (Fororderinginformation,seeRelatedProducts.)

• electrocompetent or chemically-competent cells SeeTableIII.(Fororderinginformation,seeRelatedProducts.) Note:"Oneshot"TOP10cellsarenotcompatiblewiththeCreatorCloning

System.

• Chloramphenicol stock solution (Cm) (30mg/mlin100%ethanol;1000X.)Storeat–20°C.

• Ampicillin stock solution (Amp) (100mg/mlinH2O;1000X.)Storeat–20°C.

• SoC Medium 20 g/L Bacto-tryptone 5 g/L Bacto-yeastextract 0.5 g/L NaCl Add10mlofa250mMsolutionofKCl,andadjustthepHto7.0.Autoclave.

Priortouse,add5mlof2MMgCl2(autoclaved).

• LB broth 10 g/L Bacto-tryptone 5 g/L Bacto-yeastextract 5 g/L NaCl AdjustpHto7.0with5NNaOH.Autoclave.

• LB/amp agar plates PrepareLBasdescribedabove.Priortoautoclaving,addagar(15g/L).After

autoclaving,coolto55°Candaddampicillin(100µg/ml;finalconcentration).Pourinto10-cmplatesandletharden.Theninvertplatesandstoreat4°C.

• LB/Cm/sucrose agar plates PrepareLBasdescribedabove.PriortoadjustingpH,addsucrose(7%w/v).

Addagar(15g/L)andautoclavefor20minutesmaximum(longerautoclavingtimesmaycausesucrosetoburn).Afterautoclaving,coolto55°Candaddchloramphenicol (30µg/ml).Pour into10-cmplates,and letharden.Theninvertplatesandstoreat4°C.

III. Additional Materials Required



A. Before you start • Although the reading frame of the insertion is only significant when

transferring into Acceptor Vectors that express fusion proteins, westronglyencourageyoutocloneyourgeneofinterestinframewiththeDonorVector'supstream loxPsitetoensurecompatibilitywithallofourAcceptorVectors.FordetailedDonorVectormapsandmultiplecloningsitediagrams,seeAppendicesA&B.

• WhenusingthepDNR-DualCloningKitfortheadditionof3'tagstoageneofinterest,thesequenceclonedintopDNR-DualmustbeinframewiththeSDsite,andlackstopcodonsanda3'UTRforcorrectexpres-sionoftheprotein-tagfusion.

• Crerecombinaserequiresheattoefficientlyinactivatetheenzymefol-lowingthereaction.Theenzymeretainsactivityevenwhenreactionsarestoredat4°C.

• Sucroseandchloramphenicolselectionisonlyrequiredforinitialselec-tionofcorrectrecombinants.Afterselection,recombinantclonescanbegrown in thesamemediumusedforpropagationof theAcceptorVector.

• Forbestrecombinationefficiency,werecommendusingNucleoSpin®PlasmidMinipreporMaxiprepKitstoobtainhighlypureplasmidDNA(seeRelatedProductsfororderinginformation).

• Verifyplasmidconcentrationbygelelectrophoresisbeforeperformingtherecombinationreaction.Pleasenotethatspectrophotometerread-ingscanbeinaccurateinparticularwhenRNAiscolumnpurified.

• PleaseseeAppendixCforalistofrecommendedcompetentcells. • Westronglyrecommendthatyouperformacontrolreactionusingthe

ControlDonorVectorprovidedineachkit.Performingacontrolreactionwillverifythatyoursystemisworkingproperly.

B. Creator™ DNA Cloning Procedure 1.SubcloneyourgeneofinterestintotheDonorVectorusingstandard

methods.(Formoreinformation,consultSambrooket al.,2001.) 2.PreparetheCreatorreactionmixtureasfollows: 200 ng Donor Vector 200 ng Acceptor Vector 2 µl 10X Cre Reaction Buffer 2 µl 10X BSA(1mg/ml) 1 µl Cre Recombinase

AdddeionizedH2Otobringvolumeupto20µl.Notes:

•We recommendperformingaparallel control reactionwith theappropriateControlVectorasdescribedabove(SectionA).

IV. Creator™ DNA Cloning Protocol



IV. Creator™ DNA Cloning Protocol continued

•ThefirsttimeyouuseCreRecombinase,thawrapidlyfrom–70°Cbyholdingthetubebetweenyourfingersuntilalmostalloftheiceismelted.Thenplacethetubeonice.

•Oncethawed,theCreRecombinaseshouldbestoredat–20°Cinanon-frost-freefreezer.TheCresolutionwillremainaliquidandwillnotneedtobethawedpriortouse.

3.Mixwellbygentlytappingthetube.Spinbriefly. 4.Incubateatroomtemperature(22–25°C)for15min.

Note:Donotextendincubationpast15min;competingrecombinationreactions,whichdonotgeneratedesiredrecombinants,canreducetheyieldofyourdesiredrecombi-nants.

5.Stopthereactionbyheatingtubeat70°Cfor5min. 6.Transformcompetentcellswith1µlofreactionmixture.Useatleast

50µlofcompetentcellsper1µlofreactionmixture.SeeAppendixCforalistofrecommendedcompetentcellsandvolumestouse.Note:Competentcellsshouldgive>1x108cfu/µg.Ifnot,replacewithafreshsampleofcells.

7.Growcellsat37°Cfor60mininSOCmedium(orLB). 8.Plate100µloftransformationona10-cmLB-agarplatecontaining30

µg/mlchloramphenicol,and7%sucrose(w/v). 9.Centrifugeat6,000rpmfor1min. 10.Aspirateoff800µl. 11.Resuspendin100µlofSOCmedium(orLB)andplateona10-cmLB

agarplatecontaining30µg/mlchloramphenicol,and7%sucrose(w/v).Incubateovernight.Note: It is important toallow the innoculum tosoak into theplate thoroughlybeforeincubatingovernight.

12.Thenextday,theplateshouldcontainamixtureoflargercoloniesandsmaller colonies. Pick larger colonies for screening by colony PCR(Figure4).SmallercoloniestypicallycontainamixtureofDonorandAcceptorVectorDNA.Analyzeclonesbyrestrictiondigesttoconfirmyour final construct. Plasmid(s) can be further propagated in eitherchloramphenicolortheantibioticthatisappropriatefortheresistancemarkeroftheAcceptorVector.

C. Colony PCR 1.Createa0.4µMprimermastermix.50µlofthismixwillberequired

percolonyscreened.ForrecombinationsinvolvingtheacceptorvectorspLP-CMV-HAorpLP-CMV-Myc,useacombinationofprimersPCP-1andPCP-L.ForallotheracceptorvectorsusePCP-1andPCP-2.Primersequencesare:

PCP-1:GCTCACCGTCTTTCATTGCC PCP-2:TCCGCTCATGAGACAATAACC PCP-L:TGTATCTTATCATGTCTGGATC



Figure 4. Typical plating result.ArrowsindicateexamplesoflargercoloniesthataregoodchoicesforselectionandscreeningbycolonyPCR.

2.Aliquot50µlofprimermastermixtowellsofamicrotiterplateorstrip.Picklargecoloniesfromtheplateinstep12andpipetupanddownseveraltimestoresuspendthebacteria.Use25µlofthismastermixofbacteriaandprimerstoresuspendthecontentsofonewellofaSprintAdvantage96plate(Cat.No.639550).Alternatively,othermethodsfor"colonyPCR"maybeemployed.Itisimportanthowever,toaddbroth(eg.LBorSOC)containing30µg/mlChloramphenicoloranotheran-tibioticthatisappropriateforyourplasmidtotheresuspendedcolonywithintenminutes.Thiswillensuretheviabilityofantibiotic-resistantbacteria.

3.PerformthePCRandanalyzeasdescribedinthetroubleshootingguide(Noorfewcolonies>Recombinationreactionfailed:Steps3–5).

IV. Creator™ DNA Cloning Protocol continued

Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 1�


Figure 5. The Creator™ system easily generates many constructs in one day. LuciferasewastransferredintoeachoftenAcceptorVectors.Threeclonesforeachconstructwerethenscreenedbyrestrictiondigesttoconfirmtransferoftheluciferasegene.Allbuttwoofthe30coloniescontainedthecorrectrecombinantconstruct.P=parentalvector.M=marker.*Discontinuedproductsasof3/3/2005.Datashownwasobtainedpriorto3/3/2005,pLP-AcGFP1-Cisnowavailableasareplace-mentforpLP-EGFP-C1,pLP-ECFP-C1,andpLP-EYFP-C1.

V. Typical Results

Figure5showstypicalresultsusingtheCreatorDNACloningSystemtogeneratemultipleconstructs,eachcontainingatargetgene.Inseparatetubes,pDNR-LucControlVectorwascombinedwithCreRecombinaseandtendifferentAcceptorVectors.Afterabriefrecombination,DH5αcompetentcellsweretransformedwithanaliquotofreactionmixtureandrecombinantswereselected.Withonlythreecoloniesselectedfromeachtransformation,weobtainedthedesiredrecombinantscontainingluciferasefromall10reactions.Outof30coloniespickedintotal,allbuttwocontainedtheproperinsert.WiththeCreatorDNACloningKits,95–100%ofanalyzedcloneswillcontainyourdesiredrecombinantconstruct.

M 1 2 3 P 1 2 3 PA

pLP-TRE2 pLP-RevTRE

M 1 2 3 P 1 2 3 PB

pLP-LNCX pLP-IRES2-EGFP*

M 1 2 3 P 1 2 3 1 2 3C

pLP-ECFP-C1* pLP-EGFP-C1* pLP-EYFP-C1*

M 1 2 3 P 1 2 3 P 1 2 3 P D

pLP-GADT7 pLP-GBKT7 pLP-IRESneo



VI. Troubleshooting Guide

ThesimplicityoftheCreatorDNACloningSystemmakesitsusefairlystraightfor-ward.Ifyoudonotachievetypicalresults,thisguidemayhelpyoutodeterminethesourceoftheproblem.

No or few colonies

Coloniesofvaryingsize • Sometimesdistinctlargeandsmallcoloniesmaybeseen.Insuchcases,

wegenerallyfinditbesttopickthelargercolonies.SmallcoloniestendtobemixturesofrecombinantplasmidandAcceptorVector.

Lowtransformationefficiency • Check transformationefficiency.Youshouldobtain>1x108cfu/µg;

otherwiseusenew,freshcompetentcells.ToomuchCreusedinthereaction

• ToensurethatanaccuratevolumeofCrerecombinaseisaddedtothereactionmix,usea2-or10-µlpipettor.

Celltypeused • WhileingeneralthereisnospecificrequirementtouseagivenE. coli

strain, there issomevariability inoverallefficiencies indifferentcelltypes.Thus,ifyouhavelowcolonynumbersorgetcoloniesthataremixtures(seeabove),thentryingadifferentcell typecanhelp.Testbothelectro-andchemicallycompetentversionsofdifferentcelltypes(e.g.,DH10B,XL1-Blue,Fusion-Blue,orDH5α)forbestresults.

Incubationofreactionexceeded15minutes • Do not incubate Creator Reaction Mixture longer than 15 minutes.

Competingrecombinationeventscanreducetheyieldofyourdesiredrecombinants.

• Ensure that Cre recombinase is heat-killed immediately followingincubation.Wehavefoundthatthereactionisrelativelytemperatureinsensitive,andcanevenoccuronice.Forthisreason,itiscriticalthatyoucloselymonitorthereactiontimeandproceedtothenextstepintheprotocolimmediately,ratherthanletthereactionsitonice.

PoorplasmidDNAquality

Figure 6. Amplification across a recombination juncture. Thetwoprimers,PCP-1andPCP-2,primefromthechloramphenicolmarkerandprokaryoticpromoter,respectively.Sincethesetwoele-mentsareonlylinkedthroughrecombination,successfulPCRamplificationindicatesasuccessfulrecombinationreaction.

Gene of interest

Promoter

CmrloxP loxP Prok.

Promoter

PCP-2PCP-1

Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 1�


VI. Troubleshooting Guide continued

• Forbestresults,werecommendusingNucleoSpinPlasmidProductstoobtainhighlypure,miniprepplasmidDNA.Fororderinginformation,seeRelatedProducts,SectionVIII.

• ToaccuratelydetermineplasmidDNAconcentration,analyzeanaliquotofyourminiprep,alongsideaMolecularweightstandard,ona0.8–1.0%agarose/EtBrgel.Photographthegelandcomparethebandintensityoftheplasmidtothebandintensitiesofthemassstandardstoquan-tify.PleasenotethatspectrophotometerreadingscanbeinaccurateinparticularwhenDNAiscolumn-purified.

Recombinationreactionfailed 1.Totestthesuccessoftherecombinationreaction,setupaPCRreaction

mixtureusingTITANIUM™Taq(Cat.No.639208)andprimers(PCP-1andPCP-2)thatamplifyacrossarecombinationjuncture(seeFigures2and6).Pleasenote:UsePrimerPCP-LinsteadofPCP-2ifusingpLP-CMV-MycorpLP-CMV-HAasAcceptorVectors.

2.SetupthePCRreactioninaPCRtubeasfollows: Test Negative Sample Control

18.5 µl 19.5 µl PCR-GradeWater 2.5 µl 2.5 µl 10XTITANIUMTaqPCRBuffer 1 µl CreatorReactionMixture 1 µl 1 µl PCP-1Primer(10µM) 1 µl 1 µl PCP-2Primer(10µM)

0.5 µl 0.5 µl 50XdNTPMix(10mMea.ofdATP, dCTP,dGTP,dTTP) 0.5 µl 0.5 µl 50XTITANIUMTaqDNAPolymerase 25 µl 25 µl Total volume

PrimerPCP-1:5'-GCTCACCGTCTTTCATTGCC-3' PrimerPCP-2:5'-TCCGCTCATGAGACAATAACC-3'

PrimerPCP-L:5'-TGTATCTTATCATGTCTGGATC-3'

3.Commence thermal cycling using the following parameters. Thesearegeneralguidelinesforusewithhot-lidthermalcyclers;theoptimalparametersmayvarywithdifferentthermalcyclers.

•94°Cfor2min •25cycles: 94°C 15sec 58°C 30sec 72°C 30sec •72°Cfor5min

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 �0 Version No. PR631583


VI. Troubleshooting Guide continued

M–+

kb

32

0.5

1.51

←

Figure 7. Typical test results for successful recombination.

4.Transfera5-µlsampleofyourPCRreactiontoafreshtubeandadd1µlof5Xstop/loadingbuffer.Analyzeyoursample(s)byelectrophore-sisona2.0%agarose/ethidiumbromidegel,alongwithsuitableDNAsizemarkers.

5. expected results: Thereactionshouldproduceamajorfragmentof356bp.Nobandsshouldbegeneratedinthenegative(i.e.,noDNAtemplate)control.

Note:Thistestisalsoaverysimplewaytoscreencoloniesforrecombinants.

Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 �1


VII. References

Abremski,K.&Hoess,R. (1984)BacteriophageP1site-specific recombination.PurificationandpropertiesoftheCrerecombinase.J. Biol. Chem. 259:1509–1514.

CreatorDNACloning&ExpressionSystem(April2000)Clontechniques XI(2):7–11.

Sambrook,J.,Fritsch,E.F.&Maniatis,T.(2001).Molecular Cloning: A Laboratory Manual,ColdSpringHarborLaboratoryPress(ColdSpringHarbor,NY).

Sauer,B.(1994)Site-specificrecombination:developmentsandapplications.Curr. Opin. Biotechnol. 5:521–527.

For additional reading

BarabinoS.M.L.&Keller,W. (1999)Last but not least: regulatedPoly(A)Tail Formation. Cell 99:9–11.

Guo,F.,Gopaul,D.N.&VanDuyne,G.D.(1997)StructureofCrerecombinasecomplexedwithDNAinasite-specificrecombinationsynapse.Nature 389:40–46.

Lilley,D.M.J.(1997)Site-specificrecombinationcaughtintheact.Chem. Biol. 4:717–720.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 �� Version No. PR631583


VIII. Related Products

ForthelatestandmostcompletelistingofallClontechproducts,pleasevisitwww.clontech.com

Products Cat. No.

• Creator™pDNRCloningKit 631615

• Creator™SMART™PremadecDNALibraries many

• Creator™SMART™cDNALibraryConstructionKit 634903

• Creator™AcceptorVectorConstructionKit 631618

Donor Reporter Vectors

• pDNR-LacZDonorReporterVector 631612

• pDNR-SEAPDonorReporterVector 631613

Living Colors® expression Vectors

• pLP-AcGFP1-CAcceptorVector 632471

• pLPS-AcGFP1-NAcceptorVector* 632472

Mammalian expression Vectors

• pLP-IRESneoAcceptorVector 631607

• pLP-CMV-MycAcceptorVector 631603

• pLP-CMVneoAcceptorVector 631816

• pLP-CMV-HAAcceptorVector 631817

Matchmaker Vectors

• pLP-GADT7AcceptorVector 630405

• pLP-GBKT7AcceptorVector 630406

Retroviral expression Vectors

• pLP-LNCXAcceptorVector 631504

• Adeno-X™ExpressionSystem 631524

*OnlycompatiblewithgenesclonedinpDNR-DualDonorVector.



Inducible Mammalian expression Vectors & Systems

• pLP-RevTREAcceptorVector 631015

• pLP-TRE2AcceptorVector 631016

• RevTet-OffRetroviralGeneExpressionSystem 631023 featuringCreatorcloning

• RevTet-OnRetroviralGeneExpressionSystem 631024 featuringCreatorcloning

Bacterial expression Vectors & Systems

• pLP-PROTet-6xHNAcceptorVector 631201

• Creator-CompatiblePROTet-6xHNBacterialGene 631204 ExpressionSystem

Baculovirus expression Vectors & Systems

• BacPAK™BaculovirusExpressionSystem 631402

• pLP-BacPAK9AcceptorVector 631407

• pLP-BacPAK9-6xHNAcceptorVector 631408

• BacPAK6DNA(Bsu36Idigest) 631401

Competent Cells

• Fusion-Blue™CompetentCells 636700

• SuperchargeEZ10ElectrocompetentCells 636756

Plasmid Purification Kits

• NucleoSpin®PlasmidMiniprepKits 636042 635988

• NucleoSpin®Multi-8PlasmidKits 635973 635974

• NucleoSpin®Multi-8PlusPlasmidKits 635975 635976

• NucleoSpin®Multi-96PlasmidKit 636007 636005 636006

VIII. Related Products continued



Appendix A: Creator™ Donor and Control Vector Maps

Figure 8. Map of pDNR-1r Donor Vector. TheDonorVectorcontainstwoloxPsites,whichflankthe5'endoftheMCSandthe5'endofthechloramphenicolopenreadingframe(Cmr).TheDonorVectoralsocontainsthesucrasegenefromB. subtilis (SacB)forselectionofcorrectrecombinants,andanampicillinresistancegeneforpropagationandselectioninE. coli.EachAcceptorVectorcontainsaloxPsite,followedbyabacterialpromoter,whichdrivesexpressionofthechloramphenicolmarkerafterrecombination.Thegeneofinterest,oncetransferred,willbecomelinkedtothespecificexpressionelementsforwhichtheAcceptorVectorwasdesigned.Inaddition,ifthegeneofinterestisclonedinframewiththeupstreamloxPsiteintheDonorVector,itwillautomaticallybeinframewithallpeptidesintheAcceptorVectorfollowingrecombination.Sequenceanddigestinformationisavailable,andcanbedownloadedfromourwebsiteatwww.clontech.com/clontech/techinfo/vectors/.

Figure 9. Map of pDNR-1r-Luc Control Vector. ThisvectorissimilartotheDonorVector,above,excepttheluciferasegenehasbeenclonedintotheMCS.

pDNR-1r4.9 kb

pUC ori

MCS

Ampr

SacB

loxP

Cmr

(ORF)

loxP

pDNR-1r-Luc6.4 kb

pUC ori

Ampr

loxP SacB

loxP

Cmr

(ORF)

Luciferase



Figure 10. Map of pDNR-Dual Donor Vector. pDNR-Dualcontainsasplicedonor(SD)sitedirectlydownstreamoftheMultipleCloningSite(MCS).WhencombinedwithaspecializedAcceptorVectorcontainingaspliceacceptor(SA)site,arecombinantexpressionconstructisgeneratedcontaininganartificialintron(consistingofthechloramphenicolmarkerandoneloxPsite),whichissplicedoutbytheeukaryotichost'stranscriptionalmachinery.Asaresult,atranscriptiscreatedthatexpressesthetagasa3'fusiontoyourgeneofinterest.Sequenceanddigestinformationisavailable,andcanbedownloadedfromourwebsiteatwww.clontech.com/clontech/techinfo/vectors/.

Appendix A: Creator™ Vector Maps continued

Figure 11. Map of pDNR-Dual-Luc Control Vector. ThisvectorissimilartotheDonorVector,above,withtheexceptionthattheluciferaseopenreadingframehasbeenclonedintotheMCS.

pDNR-Dual4.9 kb

pUC ori

MCS

Ampr

SacB

loxP

Cmr(ORF)

loxP

SD site6xHN tag

pDNR-Dual-Luc6.4 kb

pUC ori

Ampr

loxP SacB

loxP

Cmr

(ORF)

LuciferaseSD site6xHN tag



Figure 12. Map of pDNR-CMV Donor Vector. pDNR-CMVcontainstheimmediateearlyCMVpro-moterforexpressiontestinginmammaliancellspriortogenetransfer,twoloxPsites(flankingthe5'endoftheMCS)andthe5'endofthechloramphenicolopenreadingframe(Cmr).TheDonorVectoralsocontainsthesucrasegenefromB. subtilis (SacB)forselectionofcorrectrecombinants,andanampicillinresistancegeneforpropagationandselectioninE. coli.EachAcceptorVectorcontainsaloxPsite,followedbyabacterialpromoter,whichdrivesexpressionofthechloramphenicolmarkerafterrecombination.Sequenceanddigestinformationisavailable,andcanbedownloadedfromourwebsiteatwww.clontech.com/clontech/techinfo/vectors/.

Appendix A: Creator™ Vector Maps continued

Figure 13. Map of pDNR-CMV-LacZ Control Vector. ThisvectorissimilartothepDNR-CMVDonorVector,shownabove,withtheexceptionthatthelacZgenehasbeenclonedintotheMCS.

pDNR-CMV5.6 kb

pUC ori

MCS (686–770)

Ampr

SacB

loxP

Cmr(ORF)

loxP

Rsr II(1686)

Kpn I (3330)

Nco I (354)

Nco I (1090)

PCMV IE

SV40poly A+

pDNR-CMV-LacZ9.1 kb

pUC ori

Ampr

SacB

loxP

Cmr(ORF)

loxP

Rsr II(5153)

Kpn I (6797)

Nco I (354)

Nco I (4557)

PCMV IE

SV40poly A+

LacZ

Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 ��


Figure 15. MCS of pDNR-Dual Donor Vector. Uniquerestrictionsitesareshowninbold.TheMCSisshowninframewiththeloxPsite(frame1).ThelastfournucleotidebasesoftheloxPsitecanbeseenatthelefthandsideofthemapinbold.IfthecodingsequenceforthegeneofinterestisinframewiththeupstreamloxPsiteintheDonorVector,itwillautomaticallybeinframewithany5'peptidesintheAcceptorVector.IfthecodingsequenceforthegeneofinterestisinframewiththeSDsiteintheDonorVector,itwillautomaticallybeinframewithany3'tagsintheAcceptorVector.TheApa IsiteintheMCSismethylatedandwillonlycutinadam–hoststrain.

Appendix B: Creator™ Donor Vector MCSs

Figure 14. MCS of pDNR-1r Donor Vector. Uniquerestrictionsitesareshowninbold.TheMCSisshowninframewiththeloxPsite.ThelastfournucleotidebasesoftheloxPsitecanbeseenatthelefthandsideofthemapinbold.IfthecodingsequenceforthegeneofinterestisinframewiththeupstreamloxPsiteintheDonorVector,itwillautomaticallybeinframewithallpeptidesintheAcceptorVector.

TTA TCA GTC GAC GGT ACC GGA CAT ATG CCC GGG AAT TCC TGC AGG ATC CGC TCG AGA AGC TTT CTA GAC CAT TCG TTT GGC GCG C

GG GCC CAG GTA AGT GGT CAT AAT CAT AAT CAT AAT CAT AAT CAT AAT CAC AAC TAGCCT

EcoR I BamH ISal I Kpn I Nde I Sma I Pst I Xho I

45•

Xba I BstX IHind III BssH II

Bsp120 IApa I

124•

loxP

6xHN tagSD site

STOPFrame 1STOP

Frame 3STOP

Frame 2

TTA TCA GTC GAC GGT ACC GGA CAT ATG CCC GGG AAT TCC TGC AGG ATC CGC TCG AG

A AGC TTT CTA GAC CAT TCG TTT GGC GCG CGG GCC CAG TAG GTA AGT GAA

EcoR I BamH ISal I Kpn I Nde I Sma I Pst I Xho I

45•

Xba I BstX IHind III BssH II

STOPs

Bsp120 IApa I

95•

loxP



Appendix B: Creator™ Donor Vector MCSs continued

Figure 16. MCS of pDNR-CMV Donor Vector. Uniquerestrictionsitesareshowninbold.TheMCSisshowninframewiththeloxPsite(frame1).ThelastfournucleotidebasesoftheloxPsitecanbeseenatthelefthandsideofthemapinbold.IfthecodingsequenceforthegeneofinterestisinframewiththeupstreamloxPsiteintheDonorVector,itwillautomaticallybeinframewithany5'peptidesintheAcceptorVector.

TTA TCA GTC GAC GGT ACC GGA CAT ATG CCC GGG AAT TCC TGC AGG ATC CGC TCG AGA AGC TTT CTA GAC CAT TCG TTT GGC GCG C GG GCC CAG TEcoR I BamH ISal I Nde IKpn I Sma I Pst I Xho I

686•

Xba I BstX IHind III BssH II Bsp120 IApa I

770•loxP

Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 ��


Appendix C: Competent Cells

Creator cloning results may vary depending on the competent cells used.AtClontech,wehaveusedtheCreatorSystemsuccessfullywithmanycom-merciallyavailablecompetentcells.Inourexperience,thecellslistedinTableIhaveyieldedthebestresults.

TABLe III. ReCoMMeNDeD CoMPeTeNT CeLLS

Competent Cells Volume of Cells (µl)a

Chemically Competent MaxEfficiency®DH5α(LifeTechnologiesNo.18258-012) 50 Fusion-Blue™CompetentCells(Cat.No.636700) 50 NovaBlueSingles™(NovagenNo.70181-3) 50 XL1-Blue(StratageneNo.200236) 100electrocompetent SuperchargeEZ10ElectrocompetentCells(Cat.No.636756) 40

ElectroMAXDH10B(LifeTechnologiesNo.18290-015) 50a Per1µlofrecombinationreactionmix.



Notes

Notice to Purchaser

Thisproduct is intended tobeused for researchpurposesonly. It isnot tobeused fordrugordiagnosticpurposesnorisitintendedforhumanuse.Clontechproductsmaynotberesold,modi-fiedforresale,orusedtomanufacturecommercialproductswithoutwrittenapprovalofClontechLaboratories,Inc.

TheCreator™TechnologyisbasedontheprocessofinvitroCre-LoxPrecombination.ClontechistheassigneeofU.S.PatentNos.6,410,317;6,977,165;6,838,285andotherpatentspendingcoveringCreatorvectorsandtheselectionprocessasitrelatestotheproductionofrecombinantclones.Clontechhaschosennottoexerciseitsrighttoimposelicensefeesonthein-houseuseoftheCreatorTechnology.Howeveraroyalty-bearinglicenseisrequiredoncontractservicesandsaleordistributionofclonesmadeintheCreatorSystemformat.ForinformationonusingCreatorTechnologyforcommercialpurposes,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

ForLiving Colors®Products

AcGFP1,DsRed,HcRed,AsRed,AmCyan,ZsGreen,ZsYellowandtheirvariants:

TheseproductsarethesubjectsofpendingU.S.andforeignpatents.

Not-For-ProfitEntities:Ordersmaybeplacedinthenormalmannerbycontactingyourlocalrepresen-tativeorClontechCustomerServiceat650.919.7300.Atitsdiscretion,ClontechgrantsNot-For-ProfitEntitiesanon-exclusive,royalty-free,personal,limitedlicensetousethisproductfornon-commerciallifescienceresearchuseonly.Suchlicensespecificallyexcludestherighttosellorotherwisetransferthisproduct,itscomponentsorderivativesthereoftothirdparties.NomodificationstotheproteincodingsequencemaybemadewithoutexpresswrittenpermissionfromClontech.AnyotheruseofthisproductrequiresalicensefromClontech.Forlicenseinformation,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.



For-ProfitEntitieswishingtousethisproductarerequiredtoobtainalicensefromClontech.Forlicenseinformation,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

TheCMVpromoteriscoveredunderU.S.PatentNos.5,168,062,and5,385,839assignedtotheUniversityofIowaResearchFoundation.

Practice of the two-hybrid system is covered by U.S. Patent Nos. 5,283,173, 5,468,614, and5,667,973assignedtotheResearchFoundationoftheStateUniversityofNewYork.PurchaseofanyClontechtwo-hybridreagentdoesnotimplyorconveyalicensetopracticethetwo-hybridsystemcoveredbythesepatents.CommercialentitiespurchasingthesereagentsmustobtainalicensefromtheResearchFoundationoftheStateUniversityofNewYorkbeforeusingthem.Clontechisrequiredbyitslicensingagreementtosubmitareportofallpurchasersoftwo-hybridreagentstoSUNYStonyBrook.PleasecontacttheOfficeofTechnologyLicensing&IndustryRelationsatSUNYStonyBrookforlicenseinformation(Tel:631.632.9009;Fax:631.632.1505).

UseoftheIReS sequenceiscoveredbyU.S.PatentNo.4,937,190andislimitedtousesolelyforresearchpurposes.AnyotheruseoftheIRESsequencerequiresalicensefromWisconsinAlumniResearchFoundation.

UseoftheTetracycline controllable expression systems(the"TetTechnology")iscoveredbyaseriesofpatentsincludingU.S.patentNos.5,464,758and5,814,618,whichareproprietarytoTETSystemsHoldingGmbH&Co.KG.Academicresearchinstitutionsaregrantedanautomaticlicensewiththepurchaseof thisproduct tousetheTetTechnologyonlyfor internal,academicresearchpurposes,whichlicensespecificallyexcludestherighttosell,orotherwisetransfer,theTetTechnol-ogyoritscomponentpartstothirdparties.Notwithstandingtheabove,academicandnot-forprofitresearchinstitutionswho’sresearchusingtheTetTechnologyissponsoredbyforprofitorganizations,whichshallreceiveownershiptoalldataandresultsstemmingfromthesponsoredresearch,shallneedacommerciallicenseagreementfromIPMerchandisersinordertousetheTetTechnology.Inacceptingthislicense,allusersacknowledgethattheTetTechnologyisexperimentalinnature.TETSystemsHoldingGmbH&Co.KGmakesnowarranties,expressorimpliedorofanykind,andherebydisclaimsanywarranties,representations,orguaranteesofanykindastotheTetTechnology,patents,orproducts.AllothersareinvitedtorequestalicensefromTETSystemsHoldingGmbH&Co.KGpriortopurchasingthesereagentsorusingthemforanypurpose.ClontechisrequiredbyitslicensingagreementtosubmitareportofallpurchasersoftheTet-controllableexpressionsystemtoIPMerchandisers,Inc.Forlicenseinformation,pleasecontact:

HansPeterKneubuehlTETSystemsHoldingGmbH&Co.KGImNeuenheimerFeld58269120HeidelbergGermany

Tel+4962215880400Fax+4962215880404eMail:kneubuehl@tet-systems.deoruseourelectroniclicensingrequestformviahttp://www.tetsystems.com/main_inquiry.htm

ThisproductisalsosoldunderpatentsublicenseFORRESEARCHPURPOSESONLY.LicensesforcommercialmanufactureoruseunderthispatentmaybeobtaineddirectlyfromHarvardUni-versity.

ElectroMAX™andDH5αaretrademarksofInvitrogenCorporation.

NucleoSpin®isaregisteredtrademarkofMACHEREY-NAGELGmbHandCo.,KG.

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Singles™isatrademarkandNovagen®isaregisteredtrademarkofNovagen,Inc.Clontech,ClontechLogoandallothertrademarksarepropertyofClontechLaboratories,Inc.ClontechisaTakaraBioCompany.©2006

Documents

Creator™ DNA Cloning Kits User Manual - College of …web.as.uky.edu/biology/faculty/mirabito/BIO 510 Fall 2007...human and other mammalian sources. Using a full-length sequencing