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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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Creator™ DNA Cloning Kits User Manual
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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 4 Version No. PR631583
Creator™ DNA Cloning Kits User Manual
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
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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
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I. Introduction continued
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
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I. Introduction continued
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.
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I. Introduction continued
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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I. Introduction continued
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.
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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
I. Introduction continued
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
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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.
I. Introduction continued
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 1� Version No. PR631583
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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
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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
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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
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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
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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
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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
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Creator™ DNA Cloning Kits User Manual
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�
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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
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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
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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
Creator™ DNA Cloning Kits User Manual
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.
Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 �3
Creator™ DNA Cloning Kits User Manual
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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 �4 Version No. PR631583
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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
Protocol No. PT3460-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR631583 �5
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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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 �6 Version No. PR631583
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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 ��
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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
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 �8 Version No. PR631583
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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 ��
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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.
Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3460-1 30 Version No. PR631583
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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.
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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).
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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:
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