matchmaker™ library construction & screening kits user...

Matchmaker™ Library Construction & Screening Kits User Manual

PT3955-1 (PR742237)Published 20 April 2007

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 � Version No. PR74��37


I. Introduction 4

II. ListofComponents 7

III. AdditionalMaterialsRequired 9

IV. YeastStrains 11

V. YeastVectors 14

VI. ProtocolOverview:One-HybridLibraryConstruction&Screening 16

VI. ProtocolOverview:One-HybridLibraryConstruction&Screening 17

VII. ConstructingaReporterVectorforOne-HybridAnalysis 18

VIII. ConstructingaDNA-BDFusionVectorforTwo-HybridAnalysis 19

IX. GeneratingacDNALibrary 21

X. Constructing&ScreeningOne-HybridandTwo-HybridLibraries(overview) 27

XI. Constructing&ScreeningaOne-HybridLibrary 28

XII. Constructing&ScreeningaTwo-HybridLibrary 30

ProtocolA:ScreenbyYeastMating 30

ProtocolB:ScreenbyCotransformation 35

XIII. AnalyzingPositiveInteractions 38

XIV. TroubleshootingGuide 44

XV. References 47

XVI. RelatedProducts 50

AppendixA:TypicalResultsofdscDNASynthesis 52

AppendixB:PreparationofCompetentYeastCells—LiAcMethod 53

AppendixC:One-HybridControlVectorInformation 54

AppendixD:Two-HybridControlVectorInformation 55

ListofTables

TableI. MatchmakerYeastStrainGenotypes 11TableII. MatchmakerYeastStrainPhenotypes 11TableIII. One-HybridSystemVectors 14TableIV. Two-HybridSystemVectors 15TableV. ComparisonofMatchmakerDNA-BDVectors 19TableVI. RelationshipBetweenAmountofRNAandOptimalNumberofThermalCycles 24TableVII. Set-UpforOne-HybridControlCotransformations 29TableVIII. ControlOne-HybridCotransformations:ExpectedResults 29TableIX. Set-UpforControlTwo-HybridTransformations 33TableX. Set-UpforControlTwo-HybridMatings 34TableXI. Set-UpforControlTwo-HybridCotransformations 36TableXII. ControlTwo-HybridCotransformations:ExpectedResults 37TableXIII. AssemblingMasterMixesforPCRColonyScreening 40

TableofContents

Protocol No. PT3955-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR74��37 3


TableofContentscontinued

ListofFigures

Figure1. Screeningforprotein-DNAinteractionswiththeMatchmaker One-HybridSystem. 4Figure2. Screeningforprotein-proteininteractionswiththeMatchmaker Two-HybridSystem. 4Figure3. Generalstepsofyeastone-hybridandtwo-hybridscreening. 5Figure4. ConstructingandscreeningMatchmakerOne-HybridandTwo-Hybridlibraries. 6Figure5. ReportergeneconstructsinyeaststrainsAH109andY187. 12Figure6. MatchmakerOne-HybridLibraryConstruction&Screening. 16Figure7. MatchmakerTwo-HybridLibraryConstruction&Screening. 17Figure8. Synthesisofhigh-qualitydscDNAusingSMART™technology. 21Figure9. CHROMASPINColumnandCollectionTubes. 26Figure10. ConstructingADfusionlibrariesbyrecombination-mediatedcloninginyeast. 27Figure11. ScreeninganADfusionlibraryfortwo-hybridinteractions. 32Figure12. Strategiesforanalyzingandverifyingputativepositive one-hybridandtwo-hybridinteractions. 39Figure13. Yeastmatingtoverifyprotein-protein(two-hybrid)interactions. 42Figure14. Double-strandedcDNAsynthesizedfromControlHumanPlacentaPolyA+RNA. 52Figure15. Mapofp53HIS2ControlVector. 54Figure16. MapofpGAD-Rec2-53ADControlVector. 54Figure17. MapofpGADT7-RecTADControlVector. 55Figure18. MapofpGBKT7-53DNA-BDControlVector. 55Figure19. MapofpGBKT7-LamDNA-BDControlVector. 56

NoticetoPurchaser

Clontechproductsaretobeusedforresearchpurposesonly.Theymaynotbeusedforanyotherpurpose,including,butnotlimitedto,useindrugs, in vitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.

Practiceofthetwo-hybridsystemiscoveredbyU.S.PatentNos.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).

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™Technologyforcommercialpurposes,pleasecontactalicensingrepresentativebyphoneat650.919.7320orbye-mailatlicensing@clontech.com.

ThepBridgeVectoristhepropertyoftheInstitutNationaldelaSantéetdelaRechercheMédicale(INSERM).InquiriesregardingthecommercialuseorresaleofthisvectormustbedirectedtoINSERM,France.

NucleoSpin®andNucleoBond®arearegisteredtrademarksofMacherey-NagelGmbH&Co.

Parafilm®isaregisteredtrademarkoftheAmericanNationalCanCo.

Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc. ClontechisaTakaraBioCompany.©2006

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 4 Version No. PR74��37


Matchmaker™LibraryConstruction&ScreeningKitsprovideasimplemethodforconstructingcDNAlibrariesforyeasttwo-hybridandone-hybridscreening.ThesekitscombineMatchmakerSystemswithSMARTcDNASynthesistechnology,whichallowsyoutoconstructcDNAlibrariesfromanytissuesourcestartingwithaslittleas1µgofpolyA+RNAortotalRNA.Followingaroutinein vivo cloningstep,youcanthenscreentheselibrariesforone-hybridandtwo-hybridinteractionsusingasensitivetranscriptionalassayprovidedbyourMatchmakerSystems.

Principleoftheone-hybridassay—aprotein-DNAinteractionassay

One-hybridassaysenableyoutoidentifyandcharacterizeproteinsthatbindtoatarget,cis-actingDNAsequence—anupstreamelementthatenhancestranscriptionfromadownstreamminimalpromoter.TheassaymayalsobeusedtomaptheDNA-bindingdomainofpreviouslyknown,ornewlyidentified,DNA-bindingproteins.WiththeMatchmakerOne-HybridSystem,youcanreadilyobtainthegenesencodingthecorrespondingDNA-bindingprotein.

InaMatchmakerone-hybridassay,potentialDNA-bindingproteinsareexpressedasfusionstotheGAL4activationdomain(AD)bycloningthecorrespondingcDNAintopGADT7-Rec2,alow-copyvectordesignedforone-hybridscreening.OneormoretandemcopiesofthetargetDNAsequenceareclonedintopHIS2.1,areportervectorthatcontainsthenutritionalreportergeneHIS3.InteractionbetweenaDNA-bindingproteinandthetargetsequencestimulatestranscriptionofHIS3 (Figure1),enablingtheyeasthoststrain,Y187,aHisauxotroph,togrowonminimalmedialackinghistidine.

Principleofthetwo-hybridassay—aprotein-proteininteractionassay

Two-hybridassayscanbeusedtoidentifynovelprotein-proteininteractions,confirmsuspectedinteractions,anddefineinteractingdomains.InaMatchmakerTwo-Hybridassay,abaitgeneisexpressedasafusiontotheGAL4DNA-bindingdomain(DNA-BD),whileanothergeneorcDNAisexpressedasafusiontotheGAL4activationdomain(AD;Fields&Song,1989;Chienet al.,1991).WhenbaitandlibraryfusionproteinsinteractinayeastreporterstrainsuchasAH109,theDNA-BDandADarebroughtintoproximityandactivatetranscriptionofthereportergenes:ADE2, HIS3, lacZ, andMEL1 (Figure2).

DNA-BDandADfusionsarecreatedbycloningcDNAsintotheyeastexpressionvectorspGBKT7andpGADT7-Rec.pGBKT7expressesproteinsasfusionstotheGAL4DNA-BD,whilepGADT7-RecexpressesproteinsasfusionstotheGAL4AD.Inyeast,bothfusionsareexpressedathighlevelsfromtheconstitutiveADH1promoter(PADH1).OtherGAL4-basedDNA-BDcloningvectorssuchaspGBT9,pAS2-1,andpBridgearealsocompatiblewiththiskit.pBridgeVectorcanbeusedtoperformthree-hybridassaystoidentifyternaryproteincomplexes.

Biologicalbasisforone-hybridandtwo-hybridassays

One-hybrid(andtwo-hybrid)methodsarebasedonthefindingthatmanyeukaryotictranscription factors are modular; theirtranscriptionactivatingandDNA-bindingdomainsarestructurallyandfunctionallydistinct.This allows researchers to con-struct various gene fusions that, whenexpressed as fusion proteins in yeast,cansimultaneouslybindtoatargetDNAsequenceandactivate transcriptionofadownstream reporter (Figures 1 and 2).Matchmakersystemsusethetranscriptionactivating and DNA-binding domains ofGAL4,awell-characterizedyeasttranscrip-tion factor.To learn more about GAL4-basedyeasthybridtechnology,seeZhu,L.&Hannon,G.J.,2000.

Figure1.Screeningforprotein-DNAinteractionswiththeMatchmakerOne-HybridSystem.Inthisconstruct,threecopiesoftheDNAtarget(T)havebeeninsertedintothepHIS2.1reportervector.

GAL UAS minimal promoter ADE2, HIS3, lacZ, and MEL1

transcriptionLibrary protein

GAL4 AD

Bait protein

GAL4 DNA-BD

Figure2.Screeningforprotein-proteininteractionswiththeMatchmakerTwo-HybridSystem.

GAL4 AD

Libraryprotein

HIS3T TT minimal promoter

Transcription activator

transcription

I. Introduction



I. Introductioncontinued

ConstructingandscreeningMatchmakerone-hybridandtwo-hybridlibraries

ConstructingandscreeningMatchmakerone-hybridandtwo-hybridlibrariesconsistsoffourmainsteps(Figure3).Noticethatbothproceduresfollowthesamegeneralpath.

Ifyouintendtoscreenfortwo-hybridinteractions,thefirststep(Step1)istoconstructaDNA-BDfusionvector.If,ontheotherhand,youintendtoscreenforone-hybridinteractions,yourfirststepistoconstructaDNAtarget-HIS3 reportervector.Next(Step2),usetheSMARTreagentsweprovidetogenerateacDNAlibraryfromthepolyA+ortotalRNAthatyouprovide.

Inthecaseofyeasttwo-hybridscreening,youmayskipRNAisolation,cDNAsynthesis,andADfusionlibraryconstruction(Step3)if,insteadofpreparingyourownlibrary,youintendtoscreenoneofourmanypremadeMatchmakercDNAlibraries.Representingabroadrangeoftissues,theselibrariesareavailableasglycerolstocksorpretransformedinyeaststrainY187.WealsoofferaMatchmakerCustomLibraryService.Tousethisservice,sendusthetissueorcellsyouwishtoscreen,andwewillmaketheADfusionlibraryforyou.Pleasenote,however,thatmanyofourpremadeandpretransformedMatchmakercDNALibrariesareconstructedinhigh-copyyeastexpressionvectors,idealfortwo-hybridwork,butlesssuitableforone-hybridanalysis.WerecommendusinglowcopyvectorssuchaspGADT7-Rec2andpHIS2.1forone-hybridscreeningbecausetheygeneratefewerfalsepositives.

FollowingcDNAsynthesis,constructaGAL4ADfusionlibrarybycloningthecDNAintooneofourMatchmakerADCloningVectors:pGADT7-Rec2ifyouareconstructingaone-hybridlibrary;pGADT7-Rec if you are constructing a two-hybrid library.The cloning takes place in vivo viahomologousrecombination(Figure4).ThissteptakesadvantageofthehighlyefficientrecombinationsysteminyeasttofusedscDNAwiththeappropriateGAL4ADplasmid.Withrecombination-mediatedcloning,libraryconstructionandscreening(Steps3and4)canbecompletedinquicksuccession without the need for any bacterial transformation or amplification steps. SimplytransformyeastwiththecDNAlibraryandtheappropriateMatchmakervectors;thenspreadthecellsondropoutmediumtoselectforone-hybridortwo-hybridinteractions.

1. Construct a DNA target-reporter vector by cloning your DNA target sequence into pHIS2. Section VII

1. Construct a DNA-BD fusion vector by cloning your bait gene into pGBKT7 or other GAL4 DNA-BD vector. Section VIII

�. Generate a cDNA library from any tissue or cell source using SMART™ cDNA Synthesis technology. Section IX

One-Hybrid Library Construction & ScreeningOverview: Section VI

Two-Hybrid Library Construction & ScreeningOverview: Section VI

3. Construct a GAL4 AD fusion library using homologous recombination. Sections XI & XII

4. Screen for one-hybrid or two-hybrid interactions by yeast mating or transformation. Sections XI & XII

Figure3.Generalstepsofyeastone-hybridandtwo-hybridscreening.Formoredetailedflowchartsoftheone-hybridandtwo-hybridprocedures,pleaserefertoFigures6and7.



SMART™ cDNA synthesis and amplification by LD-PCR

ds cDNA with SMART™ III & CDS III anchors

Plate on selective mediumto screen for one-hybrid or two-hybrid interactions

Homologous recombination in yeast mediates GAL4 AD Vector assembly

Steps � & 3:Library construction(in vivocloning)~ � hours

Step 4:Screening3 days

Prepare competent yeast cells and

transform

GAL4 AD LEU2

pGADT7-Rec[�]AD Vector

Sma I-linearized

pGBKT7Two-Hybrid

DNA-BD VectorGAL4 DNA-BD

protein bait

Amp

GAL4 AD LEU2

Step 1:cDNAsynthesis~ 5 hours

TRP1

Kanr

Ampr

pHIS�One-Hybrid

Reporter Vector

DNA target

TRP1

Kanr

MCS

HIS3

pGBKT7

or

pHIS2

pGADT7-Rec[�]AD Vector

Sma I-linearized

I. Introductioncontinued

Figure 4. Constructing and screening Matchmaker One-Hybrid andTwo-Hybrid libraries. As this diagram shows,recombination-mediatedcloningmakeslibraryconstructionandscreeningquickandefficient.Thoughnotshownhere,two-hybridlibrariescanalsobescreenedbyyeastmating(SeeSectionXIIfordetails).FordetailsaboutSMARTcDNAsynthesis and amplification, please refer to Section IX. pGADT7-Rec is used for two-hybrid library construction andscreening,whilepGADT7-Rec2isusedforone-hybridlibraryconstructionandscreening.Thoughrelated,thetwovectors,denotedaspGADT7-Rec[2]inthefigure,havedifferentreplicationelements.SeeSectionXandthecorrespondingVectorInformationPacketsformoreinformation.



This kit contains sufficient reagents to make 5 one-hybrid (Cat. No. 630304) or 5 two-hybrid(Cat.No.630445)libraries.

StoreDeionizedH2O,CHROMASPINColumns,NaClSolution,Dropout(DO)Supplements,NaOAc,LiAc,PEG,TEBuffer,andYPDPlusMediumatroomtemperature.Storeyeaststrains,ControlPolyA+RNA,andtheSMARTIIIOligoat–70°C.Storeallotherreagentsat–20°C.

First-strandcDNAsynthesis• 10µl SMARTIIIOligo(10µM;5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTATGGCCGGG-3')• 10µl CDSIIIPrimer(10µM;5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30VN-3')*• 10µl CDSIII/6Primer(10µM;5'-ATTCTAGAGGCCGAGGCGGCCGACATG-NNNNNN-3')* *N=A,G,C,orT;V=A,G,orC• 20µl MMLV(MoloneyMurineLeukemiaVirus)ReverseTranscriptase• 7µl RNaseH• 100µl 5XFirst-StrandBuffer(250mMTris(pH8.3);30mMMgCl2;375mMKCl)• 100µl DTT(dithiothreitol;20mM)• 5µl ControlPolyA+RNA(HumanPlacenta;1µg/µl)• 50µl dNTPMix(dATP,dCTP,dGTP,dTTP,10mMeach)• 500µl DeionizedH2O(Cat.No.630445only)

cDNAamplification• 50µl 5'PCRPrimer(10µM;5'-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3')• 50µl 3'PCRPrimer(10µM;5'-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3')• 500µl 10XGC-MeltSolution

cDNApurification• 10 CHROMASPIN+TE-400Columns• 300µl SodiumAcetate(3M;pH4.8)

One-HybridLibraryConstruction(Cat.No.630304)• 20µg pHIS2.1ReporterVector(500ng/µl)• 20µg pGADT7-Rec2ADCloningVector(Sma I-linearized;500ng/µl)• 20µg pGAD-Rec2-53ControlVector(500ng/µl)• 20µg p53HIS2ControlVector(500ng/µl)• 0.5ml S. cerevisiae strainY187• 50ml NaClSolution(0.9%)• 10g –LeuDOSupplement• 10g –TrpDOSupplement• 10g –Leu/–TrpDOSupplement• 10g –His/–Leu/–TrpDOSupplement

Two-HybridLibraryConstruction(Cat.No.630445)• 20µg pGBKT7DNA-BDCloningVector(500ng/µl)• 25µg pGADT7-RecADCloningVector(Sma I-linearized;500ng/µl)• 20µg pGBKT7-53ControlVector(500ng/µl)• 20µg pGBKT7-LamControlVector(500ng/µl)• 20µl SV40Large-TPCRFragment(25ng/µl)• 0.5ml S. cerevisiae strainAH109

II. ListofComponents



II. ListofComponentscontinued

• 0.5ml S. cerevisiae strainY187• 50ml NaClSolution(0.9%)• 10g –LeuDOSupplement• 10g –Leu/–TrpDOSupplement• 10g –Ade/–His/–Leu/–TrpDOSupplement

YeastmakerYeastTransformationSystem2(Cat.No.630439)includesthefollowing:• 50ml 1MLiAc(10X)• 50ml 10XTEBuffer• 50ml YPDPlusLiquidMedium• 20µl pGBT9(0.1µg/µl;controlplasmid)• 2x1ml HerringTestesCarrierDNA,denatured(10mg/ml)• 2x50ml 50%PEG3350



Thefollowingreagentsarerequiredbutnotsupplied.Storeallreagentsandsolutionsatroomtemperature(20–22°C)unlessspecifiedotherwise.

First-strandcDNAsynthesisandSMART™PCRcDNAamplification• Advantage®2PCRKit(Cat.Nos.639206&639207)• Sterile,0.5-mlmicrocentrifugetubes• PolyA+ortotalRNA• Mineraloil• ThermalCycler Note:Thecyclingparametersinthisprotocolweresetusingahot-lidthermalcyclerandmaynotbeoptimalfor

nonhot-lidcyclers.

• DNAsizemarkers(1-kbDNAladder)

• 1.2%Agarose/EtBrgel

cDNAsizefractionation• 1.5-mlsterilemicrocentrifugetubes• Ring-standwithsmallclampforholdingCHROMASPINcolumns• 95%ethanol(–20°C)• 1%xylenecyanol

Constructingbaitplasmids• CompetentE. colicells.UseageneralpurposestrainsuchasDH5αorFusion-Blue

CompetentCells(Cat.No.636700) Fusion-BlueCompetentCellsareanE. coli K-12strainthatprovideshightransformation

efficiency paired with blue-white screening capability when used with the appropriateplasmids.ThestraincarriesrecAandendAmutationsthatmakeitagoodhostforobtaininghighyieldsofplasmidDNA.

• T4DNAligase• 10XT4ligationbuffer(Sambrooket al.,1989;orthebufferprovidedwiththeenzyme)• LB/ampplates• 50mMNaCl• MaterialsforpurifyingplasmidfromE. colitransformants

Yeasttransformation(Preparereagentsinsterilecontainers)• PEG/LiAcSolution(polyethyleneglycol3350/lithiumacetate) Prepareafresh10-mlsolutionjustpriortotransformationusingthestocksolutionsprovided:

Mix8mlof50%PEG3350with1mlof10XTEBufferand1mlof1MLiAc(10X).• 1.1XTE/LiAcSolution Shouldbefreshlypreparedbeforeeachtransformationusingthestocksolutionsprovided:

Combine1.1mlof10XTEwith1.1mlof1MLiAc(10X).Bringthetotalvolumeto10mlusingsterile,deionizedH2O.

• DimethylSulfoxide(DMSO;SigmaCat.No.D8418)

III. AdditionalMaterialsRequired



Yeastculture&mating• YPDMediumCat.No.630409;orprepareyourown;seetheYeastProtocolsHandbook

(PT3024-1)• YPDAmedium(YPDmediumsupplementedwith30mg/Ladeninehemisulfate;seethe

YeastProtocolsHandbook)• TEbufferorsterile,distilledH2O• Appropriatesteriletubesorflasksfortransformations• 100-and150-mmcultureplates• Sterileglassrod,bentpasteurpipette,or5-mmglassbeadsforspreadingcellsonplates• X-α-Gal(Cat.No.630407)forblue/whitescreeningofyeasttwo-hybridsexpressingMEL1 (α-galactosidase)• MinimalSDBasewithandwithoutagar(Cat.Nos.630412and630411)• 3-amino-1,2,4-triazole(3-AT;SigmaCat.No.A8056;forsuppressingbackgroundgrowth

onSDminimalmedialackingHis)• Kanamycinstocksolution(50mg/mlinH2O;1000X);Storeat–20°C.

• Ampicillinstocksolution(50mg/mlinH2O;1000X);Storeat–20°C.

Long-termlibrarystorage• 100%Glycerol• FreezingMedium:YPDmediumwith25%(v/v)glycerol

Two-HybridLibraryConstruction&ScreeningThe following dropout (DO) supplements are not supplied with the Matchmaker LibraryConstruction&ScreeningKit(Cat.No.630445).YoumustobtainthesesupplementsseparatelyfromacommercialsupplierorpreparethemyourselfusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).• –TrpDOSupplement RequiredforselectionofMatchmakerDNA-BDcloningvectorsinyeast• –His/–Leu/–TrpDOSupplement Optional triple-dropout supplement for low stringency screening of yeast two-hybrid

libraries

• –His/–TrpDOSupplement RequiredfortestingyeaststrainstransformedwithaDNA-BDplasmidforbackgroundgrowth

onSDminimalmedialackingHis

• –Ade/–TrpDOSupplement RequiredfortestingyeaststrainstransformedwithaDNA-BDplasmidforbackgroundgrowth

onSDminimalmedialackingAde

One-HybridLibraryConstruction&ScreeningThefollowingdropout(DO)supplementisnotsuppliedwiththeMatchmakerOne-HybridLibraryConstruction&ScreeningKit(Cat.No.630304).YoumustobtainthissupplementseparatelyfromacommercialsupplierorprepareityourselfusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).• –His/–TrpDOSupplement RequiredfortestingyeaststrainstransformedwithapHIS2.1reporterplasmidforbackground

growthonSDminimalmedialackingHis

PCRColony-Screening• MatchmakerADLD-InsertScreeningAmplimerSet(Cat.No.630433)

III. AdditionalMaterialsRequiredcontinued



Foradditionalinformationonthegrowthandmaintenanceofyeast,seetheYeastProtocolsHandbook(PT3024-1).WealsorecommendGuthrie&Fink’sGuide to Yeast Genetics and Molecular Biology (1991)andHeslot&Gaillardin’sMolecular Biology and Genetic Engineering of Yeasts (1992).

A. Genotypes

table i. matchmaker yeast strain genotypes

Strain Genotypea References

AH109b MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, Jameset al.,1996; gal4Δ,gal80Δ, LYS2 : : GAL1UAS-GAL1TATA-HIS3, Ourunpublished GAL2UAS -GAL2TATA-ADE2, observations URA3 : : MEL1UAS-MEL1TATA-lacZ, MEL1

Y187 MATα,ura3-52,his3-200, ade2-101, trp1-901, Harperet al.,1993 leu2-3,112,gal4Δ,met –,gal80Δ, URA3 : : GAL1UAS-GAL1TATA-lacZ, MEL1

a TheGAL1,GAL2,andMEL1upstreamactivatingsequences(UASs)areresponsivetotheGAL4transcriptionalactivator.Thetrp1, his3, gal4, and gal80mutationsarealldeletions;leu2-3, 112isadoublemutation.

b AH109isaderivativeofstrainPJ69-2AandincludestheADE2 andHIS3 nutritionalmarkersandanendogenousMEL1 gene(Jameset al.,1996).ThelacZ reportergenewasintroducedintoPJ69-2AtocreatestrainAH109.

B. Phenotypes ItisimportanttoverifythephenotypesoftheAH109andY187strains(TableII).

1.Torecoverstrainsfromfrozenstock,scrapeasmallamountofcellsfromthesurfacewithasterilelooporwoodenstickandstreakthemontoYPDAplates.

2.Incubateplatesat30°Cfor3–5daysuntilcoloniesappear.Propagateadditionalculturesonlyfromisolatedcoloniesonthisworkingstockplate.

Notes: • AH109(andtransformantsderivedfromthisstrain)shouldbemaintainedonadenine-supplementedYPD

(i.e.,YPDA)foroptimalviabilityofthestrainandtopreventselectionofspontaneousade1 orade5 mutations(Guthrie&Fink,1991).

• Ifyoucannotrecoverthestrainbyscrapingthefrozenstock,thecellsmayhavesettledtothebottomofthetubebeforethestockwasfrozen.Ifthishappens,thawthefrozencultureoniceandvortexitbeforerestreaking.

• Althoughnonlibrarystockculturesmaybethawedandrefrozenseveraltimeswithoutsignificantlydecreasingtheviability,werecommendthatyoudividetheonce-thawedstockintoaliquotsbeforeyourefreezeit.Thiswillkeeptheviabilityhigherandwillreducetheriskofbacterialcontamination.

3.TestforthenutritionalrequirementsshowninTableII. a. Usingasterilelooportoothpick,streak3–4coloniesfromtheworkingstockonto

separate,appropriateSDselectionplates. b. Incubateplatesat30°Cfor4–6days.YeastgrowssloweronSDselectionmedium

thanonYPDA. c. CompareyourresultswiththoseshowninTableII.ProceedonlyifAH109andY187

havetheexpectedphenotypes. 4.Usewell-isolatedcoloniesfromtheverifiedworkingstockplatetoinoculateliquidcultures

formatingorforpreparingcompetentcells.SealtheverifiedworkingstockplatewithParafilmandstoreat4°C.table ii. matchmaker yeast strain phenotypes

table ii. matchmaker yeast strain phenotypes

Strain SD/–Ade SD/–Met SD/–Trp SD/–Leu SD/–His SD/–Ura YPDA

AH109 – + – – – + +

Y187 – – – – – + +

Notes:

• AH109andY187cangrowonSD/–Leu/–TrpiffunctionalTRP1 andLEU2genesareintroduced.• AH109 andAH109/Y187 diploids can grow on SD/–Ade/–His if the ADE2 and HIS3 genes—carried byAH109—are

activated(i.e.,inthepresenceofGAL4).

IV. YeastStrains

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 1� Version No. PR74��37


C. Matingtypecompatibilities • Y187(MATα)canmatewithAH109,HF7c,CG-1945,Y190,orSFY526(allMATa).

D. ColonyColorandSize • Y187carriestheade2-101mutationandAH109exhibitstheAde–phenotypeintheabsenceof

GAL4.Onmediumwithlowamountsofadenine,thecolonieswillturnpinkafterafewdaysandmayturndarkerasthecolonyages.WhengrownonAde-supplementedmedium,thecolorchangemaynotbenoticeable.Thesecoloniesgrowto>2mmindiameter.However,small(



F. LeakyHIS3 expression • 3-amino-1,2,4-triazole(3-AT)isacompetitiveinhibitoroftheyeastHIS3 protein(His3p).

3-ATisusedtoinhibitlowlevelsofHis3pexpressedinaleakymannerandthustosuppressbackgroundgrowthonSDmediumlackinghistidine(Fields,1993;Durfeeet al.,1993).

• TransformantsderivedfromAH109mayshowslightlyelevatedHIS3 expressionbecauseofintrinsicDNA-bindingpropertiesofthebaitprotein.Asmallamountof3-ATisgenerallysufficienttosuppressbackgroundgrowthonSD/–His.However,ifyouareselectingforbothHIS3andADE2expression,itisgenerallynotnecessarytosuppressHIS3leakinessintheinitiallibraryscreen.

• SomeyeaststrainshaverelativelyhighbasallevelsofHis3p.IfyouuseY190(MATa)asahoststrain,25–45mM3-ATwillberequiredinthemediumtosuppressbackgroundgrowth.

• Tooptimizethe3-ATconcentrationinyourselectionmedium:

Beforestartingthisprocedure,notethat–His/–TrpDropoutSupplementisnotsuppliedwiththiskit.Youmustpurchase–His/–TrpdropoutsupplementseparatelyorprepareyourownusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).

1. Plate yeast transformants on a series of SD/–His/–Trp plates containing differentconcentrationsof3-AT.

• IfyouareworkingwithAH109transformantscontainingDNA-BDplasmidssuchaspGBKT7,werecommendyoustartbytesting[3-AT]intherange0to15mM(e.g.,0,2.5,5,7.5,10,12.5,and15mM).

• IfyouareworkingwithY187transformantscontainingpHIS2.1reporterplasmids,werecommendyoustartbytesting[3-AT]intherange10to60mM.

Note:Thesearerecommendationsonly.Theoptimalconcentrationmaybeslightlyhigherorlowerdependingontheconstructandstrainused.

2. Usethelowestconcentrationof3-ATthat,afteroneweek,allowsonlysmall(



A. One-HybridSystem 1.CloningVectors • pHIS2.1isaone-hybridreportervectorthatcontainstheHIS3 nutritionalreporter

gene.Ithasamultiplecloningsite(MCS)upstreamoftheHIS3 reportergenesothatacis-actingDNAtargetsequencecanbeinserted,andtherefore,linkedtotheminimalpromoteroftheHIS3 locus(PminHIS3).ItalsocontainsaCEN6/ARS4 sequenceforstable,low-copypropagationinyeast.

• pGADT7-Rec2isacloningvectorthatcanbeusedtoexpressaproteinofinterestasafusionwiththeGAL4activationdomain(AD).Thisvectorisengineeredfor homologousrecombination-mediatedcloninginyeast(Figure4).Thus,youcanconstructcDNA/ADfusionlibrariesbytransformingyeastwithSma I-linearizedpGADT7-Rec2(provided)anddscDNAgeneratedwiththeSMARTlibraryconstructionprotocol(SectionIX).

2.ControlVectors(AppendixC) • p53HIS2isapositivecontrolreportervectorthatcontainsthreetandemcopiesofthe

cis-actingDNAconsensussequencerecognizedbyp53.p53HIS2wasconstructedbyinsertingtheDNAtargetsintothemultiplecloningsiteofpHIS2.1.Asaresult,theDNAtargetsarepositionedjustupstreamoftheminimalpromoteroftheHIS3locus(PminHIS3)andtheHIS3 reportergene.

• pGAD-Rec2-53isapositivecontrolvectorthatencodesmurinep53asafusionwiththeGAL4AD.Yeastcellsthatcontainbothp53HIS2andpGAD-Rec2-53shouldgrowonminimalSDmedialackinghistidine—i.e.,onSD/–His/–Leu/–Trp.

TABLEIII.ONE-HYBRIDSYSTEMVECTORS

Use Epitopea YeastselectionbBacterialselectionc

Cloningvectors pHIS2.1 LinkanyDNAtargettoHIS3 TRP1 kanamycin pGADT7-Rec2 ExpresspotentialDNA-binding (Sma I-linearized) proteinsasADfusions HA LEU2 ampicillin

Controlvectors p53HIS2b DetectDNA-bindingactivity TRP1 kanamycin ofp53 pGAD-Rec2-53 Expressp53asanADfusion HA LEU2 ampicillina HA=hemagglutinin;Theseepitopetagscanbeusedtoverifyprotein-proteininteractionsin vitrobycoimmunoprecipitation

(Co-IP)usingtheantibodiesandprotocolprovidedwiththeMatchmakerCo-IPKit(Cat.No.630449).Theyarenotintendedtobeusedfordetection,affinitypurification,orCo-IPofhybridproteinsexpressedinyeast.

bThereporterandADvectorshavedifferentnutritionalmarkers,sotheycanbe independentlyselectedwhenyeasttransformantsareplatedonSDminimalmediumlackingspecificnutrients.Theselectionmediumyouchoosedependsonwhichplasmidsyouareusing,whetheryouareselectingforoneortwoplasmids,andwhetheryouareselectingforcoloniesinwhichonehybridinteractionsareoccurring.

c ThevectorscarrydifferentantibioticmarkerssothattheycanbeindependentlyselectedinE. coli.

V.YeastVectors



B Two-HybridSystem 1.CloningVectors • pGBKT7:UsedtoexpressaproteinofinterestasafusionwiththeGAL4DNAbinding

domain(DNA-BD). • pGADT7-Rec:UsedtoexpressaproteinofinterestasafusionwiththeGAL4activation

domain (AD).Thisvector isengineered for homologousrecombination-mediatedcloning.pGADT7-RecisprovidedasSma I-digestedlinearDNA.

2.ControlVectors a. PositiveControl • pGBKT7-53encodesafusionbetweentheGAL4DNA-BDandmurinep53. • SV40LargeTPCRFragmentencodesSV40largeT-antigen.UsethisDNAfragment

togetherwithpGADT7-Rectocheckthetransformation-recombinationefficiencyinyeast.In vivo,SV40LargeTPCRFragmentandpGADT7-RecrecombinetoformpGADT7-RecT, which encodes a fusion between the GAL4 AD and largeT-antigen.

• p53andSV40largeT-antigeninteractinayeasttwo-hybridassay(Li&Fields,1993;Iwabuchiet al.,1993).

b. NegativeControl • pGBKT7-Lamencodesa fusionof theGAL4DNA-BDwithhumanlaminCand

providesacontrolforafortuitousinteractionbetweenanunrelatedproteinandeitherthepGADT7-RecTcontroloryourAD/libraryplasmid.LaminCneitherformscomplexesnorinteractswithmostotherproteins(Bartelet al.,1993b;S.Fields,pers.comm.;Ye&Worman,1995).

TABLEIV.TwO-HYBRIDSYSTEMVECTORS

Use Epitopea Yeastselectionc Bacterialselectiond

Cloningvectors pGBKT7 Expressanyprotein c-Myc TRP1 kanamycin asaGAL4DNA-BDfusion pGADT7-Rec Expressanyproteinasa HA LEU2 ampicillin (Sma I-linearized) GAL4ADfusion

Controlvectors pGADT7-RecT b ExpressSV40largeT-antigen HA LEU2 ampicillin asaGAL4ADfusion pGBKT7-53 Expressp53asaGAL4 c-Myc TRP1 kanamycin DNA-BDfusion pGBKT7-Lam ExpresslaminCasaGAL4 c-Myc TRP1 kanamycin DNA-BDfusion

a HA=hemagglutinin;Theseepitopetagscanbeusedtoverifyprotein-proteininteractionsin vitrobycoimmunoprecipitation(Co-IP)usingtheantibodiesandprotocolprovidedwiththeMatchmakerCo-IPKit(Cat.No.630449).Theyarenotintendedtobeusedfordetection,affinitypurification,orCo-IPofhybridproteinsexpressedinyeast.

b Createdbyhomologousrecombinationin vivo bycotransformingyeastwithSV40LargeTPCRFragmentandpGADT7-Rec.

c TheDNA-BDandADvectorshavedifferentnutritionalmarkers,sotheycanbeindependentlyselectedwhenyeasttransformantsareplatedonSDminimalmediumlackingspecificnutrients.Theselectionmediumyouchoosedependsonwhichplasmidsyouareusing,whetheryouareselectingforoneortwoplasmids,andwhetheryouareselectingforcoloniesinwhichtwohybridproteinsareinteracting.

d ThevectorscarrydifferentantibioticmarkerssothattheycanbeindependentlyselectedinE. coli..

V.YeastVectorscontinued



Figure6.MatchmakerOne-HybridLibraryConstruction&Screening.

First-strand synthesis coupled with (dC) tailing by RT

Amplificationby LD-PCR

Poly A+ RNA

poly A 3'

SMART™ IIIOligonucleotide

CDS III oligo(dT) or random primer

Template switching and extension by RT

poly A

poly A


GGG5'CCC

5'

GGG5' CCC

VI.ProtocolOverview:One-HybridLibraryConstruction&Screening



100 ng of total RNA or 25 ng of poly A+ RNA

Amplify ds cDNA by LD-PCR.

Library Construction [One-Step]

Synthesize first-strand cDNA.

Select for transformants expressing interacting proteins.

Cotransform yeast strain AH109 with: • ds cDNA • pGADT7-Rec • DNA-BD/bait plasmid

Purify (size-select) ds cDNA.

Generate a cDNA LibrarySection IX

AnalysisVerify positive interactions.

Cotransform yeast strain AH109 with: • ds cDNA • pGADT7-Rec

Select transformants on SD/–Leu.

AnalysisVerify positive interactions.

Harvest (Pool) Transformants.

Mate with Y187 pretransformed with DNA-BD/bait plasmid.

Select for yeast diploids expressing interacting proteins.

Active or Toxic

Construct a DNA-BD FusionSection VII

Transform AH109 and Y187 with bait plasmid.Test for autonomous reporter gene activation

and cell toxicity.

See Troubleshooting Guide.

Two-Hybrid Screening

Protocol A• Construct an AD Fusion Library• Screen by Yeast Mating Section XII

Clone bait gene into pGBKT7 or other Matchmaker GAL4-based DNA-BD vector.

Inactive and Nontoxic

Two-Hybrid Screening

Prepare Y187 cells for mating.

Library Construction [Three-Step]

Protocol B• Construct an AD Fusion Library• Screen by Cotransformation Section XIIor

or

Prepare competent AH109 yeast cells

Appendix B

Prepare competent yeast cellsAppendix B

Figure7.MatchmakerLibraryConstruction&ScreeningKit.Two-hybridlibrariesmaybescreenedbyeitheryeastmatingorcotransformation.

VI.ProtocolOverview:Two-HybridLibraryConstruction&Screening



A. Background

To use the Matchmaker One-Hybrid System to screen a cDNA library for DNA-bindingproteins,youmusthaveidentifiedatrueorputativetargetelement.Itmustbepreciselydefinedusing,forexample,deletionand/orpointmutationanalysis.Aconstructcomposedofoneormoretandemcopiesofyourtargetregulatoryelementborderedbyrestrictionsitesisthenpreparedandinsertedintothemultiplecloningsite(MCS)ofpHIS2.1.ThislinksthetargetelementtotheHIS3 reportergene.InsertingyourtargetelementmayalterthelevelofbackgroundHIS3expression.Therefore,constructsshouldbetestedforbackground(leaky)HIS3 expressionbeforeyoustartaone-hybridanalysis.BackgroundgrowthduetoleakyHIS3expressioniscontrolledbyadding3-ATtotheselectionmedium,asdescribedinSectionIV.F.

B. SynthesizeYourTargetElement

Eachtarget-reporterconstructshouldcontainatleastonecopyoftheDNAtargetelementinsertedupstreamofthereportergene.ManyearlystudiesindicatedthatthereportershouldcontainatleastthreetandemcopiesoftheDNAtarget.However,asWeiet al. (1999)havedemonstrated,asinglecopymaybesufficientinmanycases.Formoreinformationabouttargetcopynumber,seeGhoshet al.,1993.Tandemcopiesmaybegeneratedbyvariousmethods,butwehavefoundthemostconvenientandreliablemethodforgeneratingthemtobeoligonucleotidesynthesis. It works nicely because well-defined regulatory elements are usually



A. ConstructaDNA-BDFusion • YoucangenerateaGAL4DNA-BDfusiongeneifcompatiblerestrictionsitesarepresentinthe

testgeneandthecorrespondingvector(TableV).Ifnot,generatethegenefragmentbyPCRusingprimersthatcontainthedesiredrestrictionsite(Scharf,1990).ArestrictionsiteattheendofagenecanoftenbechangedintoadifferentsiteorputintoadifferentreadingframebyusingaPCRprimerthatincorporatesthedesiredmutation.Alternatively,ifyouhavealreadyclonedyourgeneintoaCreatorTMDonorVector,useCrerecombinasetotransferyourgenetopLP-GBKT7.RefertotheCreatorDNACloningKitsUserManual(PT3460-1)fordetails.

• Formoredetailedinformationoncloning,seeSambrooket al.(1989). TABLEV.COMPARISONOFMATCHMAKERDNA-BDVECTORS

DNA-BDvector Description Size ProteinExpression

pGBKT7 GAL4(1–147)DNA-BD 7.3kb High TRP1,Kanr

pBridgea GAL4(1–147)DNA-BD 6.5kb Low TRP1,Ampr

pGBT9b GAL4(1–147)DNA-BD, 5.4kb Low TRP1,Ampr

pAS2-1b GAL4(1–147)DNA-BD, 8.4kb High TRP1,Ampr, CYHs2

pLP-GBKT7c GAL4(1–147)DNA-BD, 7.5kb High TRP1,Kanr,loxP aContainstwodistinctexpressioncassettesforinvestigatingternaryproteincomplexes.bDNA-BDvectorsusedinpreviousMatchmakerTwo-HybridSystems.cCreatorAcceptorVector(LP=loxP).AcceptsageneofinterestfromanyCreatorDonorVectorandexpresses

itasaGAL4DNA-BDfusion.

1.Purifythegenefragment. Note:WerecommendtheNucleoSpinExtractionKit(Cat.No.635961)forrapidisolationofDNAfragments.

2.Digest the DNA-BD vector with the appropriate restriction enzyme(s), treat withphosphatase,andpurify.

3.Ligatetheappropriatevectorandinsert.TransformligationmixturesintoE. coli. 4.Identifyinsert-containingplasmidsbyrestrictionanalysisorPCR.

B. TesttheDNA-BDFusionforTranscriptionalActivation 1.TransformAH109andY187withthehybridconstructusingasmall-scaletransformation

protocolsuchastheonegiveninSectionXII.A.7.Platetransformantsonthefollowingmedia*: • SD/–Trp/X-α-Gal • SD/–His/–Trp/X-α-Gal • SD/–Ade/–Trp/X-α-Gal Includeanegativecontrol.Forexample,transformcellswithan"empty"DNA-BDvector. * Note:Thedropoutsupplementsrequiredtomakethesemediaarenotsuppliedwiththe

MatchmakerLibraryConstruction&ScreeningKit(Cat.No.630445).Youmustpurchasethesesupplementsseparatelyfromacommercialsupplier,orpreparethemyourselfusingtherecipegiveninAppendixCoftheYeastProtocolsHandbook(PT3024-1).

2.Analyzeresults.

• BaitproteinisinactiveifthetransformantcoloniesarewhiteanddonotgrowonSD/–His/–TrporSD/–Ade/–Trp.GotoSteps5–6.

• Baitproteinisactiveiftransformantcoloniesareblue andgrowonSD/–His/–TrporSD/–Ade/–Trp. ContinuewithSteps3–4.

3.Ifabaitstrainexhibitsbackgroundgrowthon–Hismedium,youmaybeabletoeliminate(or reduce) the background by adding 3-AT to the selection medium (Section IV.F).Alternatively,use–Ade/–His/–Leu/–Trpmediumforthelibraryscreening.

4.Ifabaitstrainexhibitsbackgroundgrowthon–Adeand–Hismedium,itwillbedifficulttousethisbaitproteininatwo-hybridlibraryscreening.SeeTroubleshootingGuide.

VIII.ConstructingaDNA-BDFusionVectorforTwo-HybridAnalysis

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 �0 Version No. PR74��37


5.[Optional]Ifaknownproteinpartnerforyourbaitproteinisavailable,useittocheckwhetheratwo-hybridinteractionisdetectablewiththisparticularDNA-BD/baitfusion.

6.[Optional]Verifyproteinexpression: • PrepareWesternblotsfromthelysateoftransformantsandprobetheblotswithantibodies

totheGAL4DNA-BD(Cat.No.630403).Useuntransformedyeastlysateasacontrol. Notes: • Usingpolyclonalantibodiesmayresultinmultiplecross-reactingbands. • The level of expression from pGBT9 or pBridge is too low to permit detection using our GAL4

DNA-BDMonoclonalAntibody.

C. TesttheDNA-BDFusionforToxicity • ComparethegrowthrateinliquidcultureofY187cellstransformedwiththe"empty"

DNA-BDvectorandcellstransformedwiththeDNA-BD/baitplasmid.Ifthebaitstraingrowsnoticeablyslower,yourDNA-BD/baitproteinmaybetoxic.

Note:WealsorecommendcheckingfortoxicityinstrainAH109.

• UseaSmall-ScaleYeastTransformationProtocol(suchastheonegiveninSectionXII.A.7)topreparetransformants.SelecttransformantsonSD/–Trp,thenprepareanovernightcultureasfollows:

1. Inoculate50mlofSD/–Trp/Kan(20µg/ml)withonelarge(2–3mm)colony. 2. Incubateat30°Covernight(16–24hr)withshakingat250–270rpm. 3. ChecktheOD600oftheculture;itshouldbe≥0.8.IftheOD600ismuchlessthan0.8,

theDNA-BDfusionmaybetoxic(seetheTroubleshootingGuide).Ifthefusiondoesnotappeartohamperyeastgrowth—i.e.isnontoxic—andyouplantoscreenyourtwo-hybridlibrarybyyeastmating,continuewithSteps4–7.Ifyouareplanningtoscreenbycotransformation,youmaystophereandproceedtoSectionIX.

4. Centrifugeat600xgfor5min. 5. Removesupernatant. 6. Resuspendin~5mlSD/–Trpliquidmedium.Countcellsusingahemacytometer.The

celldensityshouldbe≥1x109cells/ml. 7. MatethisbaitstrainwithyourADfusionlibraryhoststrain(SectionXII.A.4). Note:Y187(MATα)canmatewithAH109,HF7c,CG-1945,Y190,orSFY526(allMATa).

VIII.ConstructingaDNA-BDFusionVectorforTwo-HybridAnalysiscontinued

Protocol No. PT3955-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR74��37 �1


A. GeneratingacDNAlibrarywithSMART™technologyMessengerRNAtranscriptsareefficientlycopiedintodscDNAusingSMART(SwitchingMechanism at 5' end of RNA Transcript) technology (Zhu,Y.Y., et al., 2001).This cDNAsynthesisandamplificationsystemisparticularlywellsuitedforone-hybridandtwo-hybridlibraryconstructionbecauseitconsistentlydelivershighyieldsofcDNAwhilemaintainingsequencerepresentation.Bymaintainingthecomplexityoftheoriginaltissue,theSMARTprocedureprovidesyouwiththebestopportunityofdetectingrareandpotentiallynovelinteractionsduringyeastone-hybridandtwo-hybridscreening.

B. HowSMARTcDNASynthesisandAmplificationworksInthefirst-strandcDNAsynthesisstep,MMLV(MoloneyMurineLeukemiaVirus)ReverseTranscriptase(RT)isusedtotranscribeRNAintoDNA.ToprimeRNAforcDNAsynthesis,youmayuseeitheramodifiedoligo(dT)primer(ourCDSIIIPrimer)orarandomprimer(ourCDSIII/6Primer).ThecompositionoftheresultingcDNAlibrarymaydifferdependingonwhichprimeryouchoose.IfyouusetheCDSIIIPrimer,whichhybridizestothe3'-endofpolyA+RNA,sequencesclosetothe5'-endofthetranscriptmaybeslightlyunder-represented.IfinsteadyouusetheCDSIII/6Primer,arandomprimerthatcanhybridizetomanydifferentsequencesontheRNAtemplate,yourlibraryshouldcontainavarietyof5'-and3'-endsequences,whicharerepresentedinnearequalproportions.WhenMMLVRTencountersa5'-terminusonthetemplate,theenzyme’sterminaltransferaseactivityaddsafewadditionalnucleotides,primarilydeoxycytidine,tothe3'endofthecDNA.TheSMARTIIITMOligonucleotide,whichhasanoligo(G)sequenceatits3'end,base-pairswiththedeoxycytidinestretch,creatinganextendedtemplate(Figure8).RTthenswitchestemplatesandcontinuesreplicatingtotheendof theoligonucleotide. In themajorityofsyntheses,theresultingsscDNAcontainsthecomplete5'endofthemRNAaswellasthesequencecomplementarytotheSMARTIIIOligo,whichthenservesasauniversalprimingsite(SMARTanchor)inthesubsequentamplificationbylong-distancePCR(LDPCR;Chenchiket al., 1998).OnlythosesscDNAshavingaSMARTanchorsequenceatthe5'endcanserveasatemplateandbeexponentiallyamplifiedbylong-distancePCR(LDPCR).In the second step, ss cDNA is amplified by LD PCR to produce a ds cDNA library.Werecommend using theAdvantage® 2 PCR Kit (Cat. Nos. 639206 & 639207) to generateandamplifydscDNA.TheAdvantage2PolymeraseMixconsistsofTITANIUMTaqDNAPolymerase (anuclease-deficientN-terminaldeletionofTaqDNApolymerase), TaqStartAntibodytoprovideautomatichot-startPCR(Kellogget al.,1994),andaminoramountofaproofreadingpolymerase.ThispolymerasesystemletsyouamplifycDNA(aslargeas20kb)withafidelityratesignificantlyhigherthanthatofconventionalPCR(Barnes,1994).

First-strand synthesis coupled with (dC) tailing by RT

Amplificationby LD-PCR

Poly A+ RNA

poly A 3'

SMART™ IIIOligonucleotide

CDS III oligo(dT) or random primer

Template switching and extension by RT

poly A

poly A


GGG5'CCC

5'

GGG5' CCC

Figure8.Synthesisofhigh-qualitydscDNAusingSMARTtechnology.

IX.GeneratingacDNALibrary

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 �� Version No. PR74��37


C. GoodLaboratoryPractices • WearglovestoprotectyourRNAandcDNAsamplesfromdegradationbynucleases. • Whenresuspendingpelletsormixingreactions,gentlypipetthesolutionupanddown

ortapthebottomofthetube.Spinbrieflytobringcontentstothebottomofthetube.Donotvortexsampleswhenresuspendingpellets;vortexingmayshearyourcDNA.

• Performallreactionsonice,unlessotherwiseindicated. • Addenzymestoreactionmixtureslast.Besuretheenzymeisthoroughlyblendedinto

thereactionmixturebygentlypipettingthemixtureupanddown. • Donotincreasethesize(volume)ofanyofthereactions.Allcomponentshavebeen

optimizedforthevolumesspecified. • Ethidiumbromideisacarcinogen.Useappropriateprecautionsinhandlinganddisposing

thisreagent.Formoreinformation,seeSambrooket al.(1989).BondExEthidiumBromideDetoxificationCartridgesareavailablefromClontech.

• Phenolisacorrosive.Alwayswearglovesandprotectiveclothingwhenhandlingsolutionscontainingthisreagent.Ifpossible,handlesolutionscontainingphenoland/orchloroformunderachemicalfumehood.

• Inpreparingyour reactions,use theDeionizedH2Osupplied.Otherwise,use freshlydeionized(e.g.,Milli-Q-grade)H2O,withouttreatmentwithDEPC(diethylpyrocarbonate).AvoidusingautoclavedH2ObecauserecycledsteaminsomeautoclavescanintroducecontaminantsthatmayinterferewithPCR.

• Rinseallglasswarewith0.5NNaOH,followedbydeionizedH2O.Thenbaketheglasswareat160–180°Cfor4–9hr.

• Useonlysingle-useplasticpipettesandpipettetipswhenhandlingRNA.

D. RNAIsolation • ManyproceduresareavailablefortheisolationoftotalRNAandpolyA+RNA(Chomczynski

&Sacchi,1987;Farrell,1993;Sambrooket al.,1989).ClontechoffersseveralkitsfortheisolationoftotalRNAandsubsequentisolationofpolyA+RNA(seeRelatedProducts).

• TheminimumamountofstartingmaterialforcDNAsynthesisis100ngoftotalRNAor25ngofpolyA+RNA.Ingeneral,themoreRNAyoustartwith,thefewerPCRcycleswillberequiredforamplification(seeTableVI).FewerthermalcyclesarelesslikelytogeneratenonspecificPCRproducts,andthereforearebestforoptimalcDNAandlibraryquality.Thus,ifyourRNAsampleisnotlimiting,usethehigherstartingamountsofRNAshowninthetable.

E. RNAAnalysis • ThesequencecomplexityofthedscDNAsynthesized,andultimatelyofthecDNAlibrary

constructed,dependsonthequalityoftheexperimentalRNAstartingmaterial.Therefore,beforeyouuseitinafirst-strandsynthesis,werecommendyouestimatetheintegrityoftheRNAbyexaminingasampleonadenaturingformaldehyde/agarosegel.TotalRNAfrommammaliansourcesshouldappearastwobrightbands(28Sand18SribosomalRNA)atapproximately4.5and1.9kb.Theratioofintensitiesofthe28Sand18SrRNAbandsshouldbe1.5–2.5:1.IntactmammalianpolyA+RNAshouldappearasasmear(usually0.5–10kb[ormore])withfaint28Sand18SrRNAbands.Thesizedistributionmaybeconsiderablysmaller(0.5–3kb)fornonmammalianspecies(e.g.,plants,insects,yeast,amphibians,etc.).

• Iftheratioofintensityof28SRNAto18SRNA(fortotalRNA)islessthan1:1orifyourexperimentalpolyA+RNAappearssignificantlysmallerthanexpected(e.g.,nolargerthan1–5kb),wesuggestyoupreparefreshRNAaftercheckingyourRNApurificationreagentsforRNaseandotherimpurities.Ifproblemspersist,youmayneedtofindanothersourceoftissueorcells.

IX.GeneratingacDNALibrarycontinued



PLEASE READ ENTIRE PRoToCoL BEfoRE STARTING. • WesuggestyoualsoperformapositivecontrolcDNAsynthesiswithHumanPlacenta

PolyA+RNA.ThiscontrolletsyouverifythatallcomponentsareworkingproperlyandletsyouevaluatetheyieldandsizesofthedscDNAsynthesizedfromyourRNAsample.

• Intheprotocolsthatfollow,youhavetheoptionofprimingfirst-strandcDNAsynthesiswithanoligo(dT)orrandomprimer(SectionsFandG,respectively).Thereactionconditionsvaryslightlydependingontheprimerused.

F. SynthesizeFirst-StrandcDNAusinganOligo(dT)Primer 1.Combinethefollowingreagentsinasterile0.25-mlmicrocentrifugetube: 1–2 µl RNAsample(0.025–1.0µgpolyA+or0.10–2.0µgtotalRNA)

(Forthecontrolreaction,use1µl[1µg]ofthecontrolRNA.) 1.0 µl CDSIIIPrimer

1–2 µl DeionizedH2Otobringvolumeupto4.0µl.

4.0 µl Totalvolume

2.Mixcontentsandspinbriefly. 3.Incubateat72°Cfor2min. 4.Coolonicefor2min. 5.Spinbriefly. 6.Addthefollowingtothereactiontube: 2.0 µl 5XFirst-StrandBuffer 1.0 µl DTT(20mM) 1.0 µl dNTPMix(10mM)

1.0 µl MMLVReverseTranscriptase 9.0 µl Totalvolume

7.Mixgentlybytapping.Spinbriefly. 8.Incubateat42°Cfor10min. 9.Add1.0µlSMARTIIIOligonucleotide. 10.Incubateat42°Cfor1hrinanairincubatororhot-lidthermalcycler. Note:Ifyouuseawaterbathornonhot-lidthermalcyclerforthisincubation,coverthereactionmixturewith

onedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

11.Placethetubeat75°Cfor10mintoterminatefirst-strandsynthesis. 12.Coolthetubetoroomtemperature,thenadd1.0µlRNaseH. 13.Incubateat37°Cfor20min. 14.Ifyouplan toproceeddirectly to theLD-PCRstep, takea2-µlaliquot fromthefirst-

strandsynthesisandplaceitinaclean,prechilled,0.5-mltube.Placethetubeonice,andproceedtoSectionH.Ifyouusedmineraloilinyourfirst-strandreactiontube,besuretotakethe2-µlsamplefromthebottomofthetubetoavoidtheoil.

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

G. SynthesizeFirst-StrandcDNAusingaRandomPrimer 1.Combinethefollowingreagentsinasterile0.25-mlmicrocentrifugetube: 1–2 µl RNAsample(0.025–1.0µgpolyA+or0.10–2.0µgtotalRNA)

(Forthecontrolreaction,use1µl[1µg]ofthecontrolRNA.) 1.0 µl CDSIII/6Primer

1–2 µl DeionizedH2Otobringvolumeupto4.0µl. 4.0 µl Totalvolume

2.Mixcontentsandspinbriefly. 3.Incubateat72°Cfor2min. 4.Coolonicefor2min. 5.Spinbriefly.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 �4 Version No. PR74��37


6.Keepthetubeatroomtemperatureandaddthefollowing: 2.0µl 5XFirst-StrandBuffer 1.0µl DTT(20mM) 1.0µl dNTPMix(10mM) 1.0 µlMMLVReverseTranscriptase

9.0µl Totalvolume

7.Mixgentlybytapping.Spinbriefly. 8.Incubateat25–30°Cfor10minatroomtemperature. 9.Incubateat42°Cfor10min. 10.Add1.0µlSMARTIIIOligonucleotide. 11.Incubateat42°Cfor1hrinanairincubatororhot-lidthermalcycler. Note:Ifyouuseawaterbathornonhot-lidthermalcyclerforthisincubation,coverthereactionmixturewith

onedropofmineraloilbeforeyouclosethetube.Thiswillpreventlossofvolumeduetoevaporation.

12.Placethetubeat75°Cfor10mintoterminatefirst-strandsynthesis. 13.Coolthetubetoroomtemperature,thenadd1.0µl(2units)RNaseH. 14.Incubateat37°Cfor20min. 15.Ifyouplan toproceeddirectly to theLD-PCRstep, takea2-µlaliquot fromthefirst-

strandsynthesisandplaceitinaclean,prechilled,0.5-mltube.Placethetubeonice,andproceedtoSectionH.Ifyouusedmineraloilinyourfirst-strandreactiontube,besuretotakethe2-µlsamplefromthebottomofthetubetoavoidtheoil.

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

H. AmplifydscDNAbyLongDistancePCR(LD-PCR)TableVIshowstheoptimalnumberofthermalcyclestousebasedontheamountofRNAusedinthefirst-strandsynthesis.FewercyclesgenerallymeanfewernonspecificPCRproducts.TheoptimalcyclingparametersinTableVIweredeterminedusingtheControlPolyA+HumanPlacentaRNA;theseparametersmayvarywithdifferenttemplatesandthermalcyclers.

TABLEVI.RELATIONSHIPBETwEENAMOUNTOFRNAANDOPTIMALNUMBEROFTHERMALCYCLES

TotalRNA(µg) PolyA+RNA(µg) NumberofCycles

1.0–2.0 0.5–1.0 15–20

0.5–1.0 0.25–0.5 20–22

0.25–0.5 0.125–0.25 22–24

0.05–0.25 0.025–0.125 24–26




1.PreheatthePCRthermalcyclerto95°C. 2.TopreparesufficientdscDNAfortransformation,setuptwo100-µlPCRreactionsfor

eachexperimentalsample.SetuponereactionfortheControlsample.Ineachreactiontube,combinethefollowingcomponents:

2 µl First-StrandcDNA 70 µl DeionizedH2O 10 µl 10XAdvantage2PCRBuffer 2 µl 50XdNTPMix 2 µl 5'PCRPrimer 2 µl 3'PCRPrimer 10 µl 10XGC-MeltSolution 2 µl 50XAdvantage2PolymeraseMix

100 µl Totalvolume

3.Mixgentlybyflickingthetube.Centrifugebriefly. 4.Overlaythereactionmixturewith2dropsofmineraloilifnecessary.Capthetubeand

placeitinapreheated(95°C)thermalcycler. 5.Beginthermalcycling.Ifyouhaveahot-lidthermalcycler,usethefollowingprogram:

•95°C30sec •xcyclesa: 95°C 10sec 68°C 6minb •68°C 5min

a RefertoTableVItodeterminetheoptimalnumberofcyclestouse.

b Programthecyclertoincreasetheextensiontimeby5secwitheachsuccessivecycle.Forexample,inthesecondcycle,theextensionshouldlast6minand5sec;inthethird,6minand10sec.Andsoon.

Note:Thesecyclingparametersmaynotbeoptimalfornonhot-lidthermalcyclers. 6.Whenthecyclingiscomplete,analyzea7-µlaliquotofthePCRproductfromeachsample

alongside0.25µgofa1-kbDNAsizemarkerona1.2%agarose/EtBrgel.TypicalresultsobtainedwithHumanPlacentaPolyA+RNAareshowninAppendixA.IfyourPCRproductdoesnotappearasexpected,refertotheTroubleshootingGuide.

7.ProceedwithSectionIorstoredscDNAat–20°Cuntiluse.

I. PurifydscDNAwithaCHROMASPIN™TE-400ColumnCHROMASPINColumnsarepackedwithresinsthatfractionatemoleculesbasedonsize.Moleculeslargerthantheporesizeareexcludedfromtheresin.Thesemoleculesquicklymovethroughthegelbedwhenthecolumniscentrifuged,whilemoleculessmallerthantheporesizeareheldback.Inthefollowingprotocol,aCHROMASPINTE-400ColumnisusedtoselectforDNAmolecules>200bp.FormoreinformationaboutCHROMASPINColumns,pleaserefertotheCHROMASPINColumnsUserManual(PT1300-1),availableatourwebsiteatwww.clontech.com.Note:WerecommendcentrifugingCHROMASPINColumnsinaswingingbucketorhorizontalrotor.Fixed-anglerotorscanalsobeused,butthereisariskthataportionofthesamplewillslidedowntheinnersideofthecolumninsteadofpassingthroughthegelmatrix,resultinginreducedorinconsistentpurification.Performthefollowingstepsforeachexperimentalandcontrolsample.

1.RemovetheCHROMASPINColumnfromtheprotectiveplasticbagandinvertitseveraltimestoresuspendthegelmatrixcompletely.Useonecolumnforeach~95-µlcDNAsample.

2.HoldingtheCHROMASPINColumnupright,graspthebreak-awayendbetweenyourthumbandindexfingerandsnapoff(Figure9).Placetheendofthespincolumnintooneofthe2-mlmicrocentrifuge(collection)tubes,andliftoffthetopcap.Savethetopcapandthewhite-endcap.




3.Centrifugeat700xgfor5min.Aftercentrifugation,thecolumnmatrixwillappearsemi-dry.Thissteppurgestheequilibrationbufferfromthecolumnandre-establishesthematrixbed.

Note:Thecolumnfittedinthe2-mlmicrocentrifugetubecanbeplacedinsidea17x100-mmpolypropylenetubeduringcentrifugationinaswingingbucketrotor.

4.Removethespincolumnandcollectiontubefromthecentrifugerotor,anddiscardthecollectiontubeandcolumnequilibrationbuffer.

Note:AlwaysholdboththeCHROMASPINColumnandthe2-mlmicrocentrifugetubewhenremovingthemfromtherotor.

5.Placethespincolumnintothesecond2-mlmicrocentrifugetube.CarefullyandslowlyapplyyourcDNAsample(~95µlfromStepIX.H.7)tothecenterofthegelbed’sflatsurface.Donotallowanysampletoflowalongtheinnerwallofthecolumn.

Note:Aconventional,tapered1.5-mlmicrocentrifugetubecanbesubstitutedforthe2-mlcollectiontube.Thiswillallowthesampletobeconfinedtoanarrowerareaforeasierhandling.

6.Centrifugeat700xgfor5min. 7.Removethespincolumnandcollectiontubefromtherotoranddetachthemfromeach

other.Thepurifiedsampleisatthebottomofthecollectiontube.Note:Holdthesamplecollectiontubetopreventitfromdetachingfromthespincolumn.

8.Combineduplicateexperimentalsamplesinasingletube. 9.Addthefollowingreagents: 1/10 vol. SodiumAcetate(3M;pH4.8) 2.5 vol. 95%ethanol(–20°C) 10.Mixgentlybyrockingthetubebackandforth. 11.Placethetubeina–20°Cfreezeroradry-ice/ethanolbathfor1hr.(Optional:Youmay

incubateat–20°Covernight,whichmayresultinbetterrecovery.) 12.Centrifugethetubeat14,000rpmfor20minatroomtemperature. 13.Carefullyremovethesupernatantwithapipette.Donotdisturbthepellet. 14.Brieflycentrifugethetubetobringallremainingliquidtothebottom. 15.Carefullyremoveallliquidandallowthepellettoairdryfor~10min. 16.Resuspendthepelletin20µlofDeionizedH2Oandmixgently.ThecDNAisnowready

for in vivo recombination (Library Construction) with pGADT7-Rec or pGADT7-Rec2.ProceedwithOne-HybridorTwo-HybridLibraryConstruction,orstorethecDNAat–20°Cuntilyouareready.

CHROMA SPIN Column main body

2-ml Collection Tubes

Break-away end

White-end cap

Clear top cap

Matrix

Figure9.CHROMASPINColumnandCollectionTubes.Notethataconventional,tapered1.5-mlmicrocentrifugetubecanbesubstitutedforthe2-mlcollectiontube.Thiswillallowthesampletobeconfinedtoanarrowerareaforeasierhandling.




PT7 TADH1GAL4 AD pGADT7-Rec[�]pGADT7-Rec[�] HAPADH1

pGADT7-Rec or pGADT7-Rec� Vector (Sma I-linearized)

SMART™ III ds cDNA

Recombination

CDS III

LEU2

Figure10.ConstructingADfusionlibrariesbyrecombination-mediatedcloninginyeast.ToclonecDNAintopGADT7-RecorpGADT7-Rec2,cotransformyeastwithdscDNA(generatedwithourmodifiedSMARTprocedure)andeitherpGADT7-Rec (two-hybrid screen)orpGADT7-Rec2 (one-hybrid screen).Yeast repair enzymeswill restore theSma I-linearizedplasmidtoitscircularformbyrecombiningsequencesattheendsofthecDNAwithhomologoussequencesattheendsofpGADT7-Rec(2).TheoutcomeisafullyfunctionalGAL4AD/cDNAexpressionvector.

A. ConstructingGAL4ADFusionLibrariesforOne-HybridandTwo-HybridScreening 1.Whetheryouplantoscreenforone-hybridortwo-hybridinteractions,themethodsused

toconstructthelibraryarethesame.Inbothcases,recombination-mediatedcloningisusedtofuseSMARTdscDNAwiththeGAL4AD(Figure10).Whilethecloningmethodsarethesame,theGAL4ADcloningvectorsarenot—

• Toconstructaone-hybridlibrary,usepGADT7-Rec2. • Toconstructatwo-hybridlibrary,usepGADT7-Rec.

pGADT7-Rec2andpGADT7-Recdifferintheirmodeofreplication.pGADT7-Recisahigh-copyplasmid;itcontainsa2µoriginofreplication,whichenablesittoreplicatemultipletimesduringthecellcycle.pGADT7-Rec2,ontheotherhand,isalow-copyplasmid;itcontainsanautonomousreplicationsequence,orARSelement,whichallowsthevectortoreplicateonlyonceduringthecellcycle.pGADT7-Rec2alsocontainsacentromericsequence,orCEN element,toensurestablesegregationoftheplasmidduringmitosisandmeiosis. Low-copyplasmidssuchaspGADT7-Rec2arepreferred forone-hybridanalysisbecausetheygeneratefewerfalsepositives.

2.AGAL4ADfusionlibraryisproducedbycotransformingyeastwithSMARTdscDNAand Sma I-linearized pGADT7-Rec or pGADT7-Rec2, depending on whether you areconstructingatwo-hybridorone-hybridlibrary.SMARTdscDNArecombineswiththeADcloningvectorin vivotoyieldacompleteGAL4ADexpressionvector(Figure10).TheresultingconstructwillexpressthecDNAinsertasaGAL4ADfusionprotein.Thisone-stepcloningprocedureispossiblebecausetheSMARTIIIandCDSIIIsequences—incorporatedintothecDNAbyRTandLD-PCR—havebeenengineeredintothepGADT7-RecandpGADT7-Rec2plasmids.Inyeast,thelinearplasmidisrestoredtoitscircularformbyrecombinationwithoverlappingsequencesattheendsoftheSMARTcDNA.Successfulplasmidassemblyresultsinapositive(Leu2+)transformant.

B. ScreeningGAL4ADFusionLibraries 1.One-HybridLibraryScreening(cotransformation) Withrecombination-mediatedcloning(Figure10), libraryconstructionandscreening

can be carried out in the same host strain on the same day. If you prepare a DNAtarget/reporterplasmid—e.g.,pHIS2.1/DNAtarget—inadvance,youcanincludeitinthecotransformationreactiontogetherwithyourcDNAlibraryandpGADT7-Rec2.Withasingletransformationstep,allthreeDNAcomponentscanbeintroducedintotheyeastreporterstrain(Figure4).ScreeningbeginsassoonasthepGADT7-Rec2ADvectorisassembledbythehost’srecombinationprocesses.Positiveone-hybridscanbeidentifiedimmediatelyaftercotransformationbyplatingthetransformationmixtureonmediumthatselectsfortheHIS3nutritionalreporter.Forprotocoldetails,seeSectionXI.

2.Two-HybridLibraryScreening(yeastmatingorcotransformation) Two-Hybridlibrariescanbescreenedbyeitheryeastmatingorcotransformation.As

describedabove,cotransformationallowsyoutoconstructandscreenyourlibraryinasinglehoststrain.Theprocedureisquickandefficient.FordetailspleasereviewProtocolsAandBinSectionXIIandseetheTwo-HybridLibraryConstruction&ScreeningflowchartinFigure7.

X. Constructing&ScreeningOne-HybridandTwo-HybridLibraries



PLEASE READ ENTIRE PRoToCoL BEfoRE STARTING.Beforestartinglibraryconstruction: • PouranappropriatenumberofSDagarplates: – SD/–His/–Leu/–Trp+optimal[3-AT]toselectforone-hybridinteractions(150-mm) – SD/–Leutomeasurethetransformationefficiencyofthelibraryplasmid(100-mm) – SD/–Trptomeasurethetransformationefficiencyofthereporterplasmid(100-mm) – SD/–Leu/–Trptomeasurethenumberofclonesscreened(100-mmplates) AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hr. • PreparePEG/LiAcSolution(SectionIII). • Be sure to run the necessary controls (Part C) in parallel with your experimental

sample.

Important:Notethatyoumustpreparecompetentyeastcellsbeforestartinglibraryconstruction.PleasetakesometimetoreviewtheprocedureinAppendixB,andplanyourworkaccordingly.

A. CotransformYeastStrainY187withdscDNA,pGADT7-Rec2,andpHIS2.1/targetDNA. 1.Preparecompetentyeastcells(AppendixB). 2.Inasterile,15-mltubecombinethefollowing:

• 20 µl dscDNA(fromSectionIX.I,Step16) • 6 µl pGADT7-Rec2(0.5µg/µl) • 5 µgpHIS2.1/targetDNA(preparedinSectionVII) • 20 µl HerringTestesCarrierDNA,denatured*

Note:ThecombinedvolumeoftheseDNAcomponentsshouldnotexceed60µl,or1/10thevolumeofthecompetentcellsaddedatStep3,below.

* Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.

3.Add600µlofcompetentcellstotheDNA. 4.Gentlymixbyvortexing. 5.Add2.5mlPEG/LiAcSolution. 6.Mixgentlybyvortexingfor3–5sec. 7.Incubateat30°Cfor45min.Mixcellsevery15min. 8.Add160µlDMSO,mix,andthenplacethetubeina42°Cwaterbathfor20min.Mix

cellsevery10min. 9.Centrifugeat700xgfor5min. 10.Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium. Note:YPDPlusisspeciallyformulatedtopromotetransformation.DonotusestandardYPDmediumforthisstep.

11.Incubateat30°Cwithshakingat~265rpmfor90min. 12.Centrifugeat700xgfor5min. 13.Discardthesupernatantandresuspendin6mlofNaClSolution(0.9%).

B. SelectforOne-HybridInteractions 1.Todeterminethetransformationefficiencyandtocalculatethenumberofclonesscreened,

spread100µlofa1:10,1:100,and1:1,000dilutiononto100-mmSD/–Leu,SD/–Trp,andSD/–Leu/–Trpagarplates.

2.SpreadtheremainingmixtureonSD/–His/–Leu/–Trp+optimal[3-AT]plates(150µlcells/150-mmplate)toselectforone-hybridinteractions.

3.Incubateat30°Cfor3–7daysuntilcoloniesappear. 4.Calculatethetransformationefficiencyandnumberofclonesscreened: a. ColoniesonSD/–Leuxdilutionfactor÷volume(ml)platedx6ml=#transformantsper

3µgpGADT7-Rec2.Expected:≥1x106transformants/3µgpGADT7-Rec2 b. ColoniesonSD/–Leu/–Trpxdilutionfactor÷volume(ml)platedx6ml=#clones

screened Expected:≥3x105clones/library

XI. Constructing&ScreeningaOne-HybridLibrary



5.RestreaktheHis+coloniesonfreshSD/–His/–Leu/–Trp+optimal[3-AT].Incubateat30°Cfor2–4days.SealtheplateswithParafilmandstoreat4°C.Forlong-termstorage,prepareglycerolstocksandstoreat–70°C.

6.AnalyzepositiveinteractionsasdescribedinSectionXIII.

C. One-HybridControls

• pGAD-Rec2-53• p53HIS2

Transform

Y187Y187[pGAD-Rec2-53 + p53HIS2]

One-Hybrid Controls

Control Strain[plasmids]Cotransform Y1�7 with:

1. Positive Control

• pGAD-Rec2-53• pHIS2 Y187[pGAD-Rec2-53 + pHIS2]

�. Negative ControlTransform

Y187

TABLEVII.SET-UPFORONE-HYBRIDCONTROLCOTRANSFORMATIONS

Component #1PositiveControl(µl)#2NegativeControl(µl)

pGAD-Rec2-53(500ng/µl) 1.0 1.0p53HIS2(500ng/µl) 1.0 —pHIS2.1(500ng/µl) — 1.0HerringTestesCarrierDNA(10mg/ml),denatured* 5 5Y187CompetentYeastCells 50 50PEG/LiAcSolution 500 500

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe1.5-mlreactiontube.

1.Preparecompetentyeastcells(AppendixB). 2.Setuptwo1.5-mlmicrocentrifugetubes.AddDNA,competentyeastcells,andPEG/LiAc

Solutionusingthevolumesandintheorderindicated(TableVII). 3.Mixthoroughlybygentlyvortexing. 4.Incubateinawaterbathat30°Cfor30min.Vortexgentlyevery10min. 5.Add20µlofDMSOtoeachtube,mix,andthenplacethetubeina42°Cwaterbathfor

15min.Vortexgentlyevery7.5min. 6.Centrifugeathighspeedfor15sec. 7.Removesupernatantandresuspendin1mlofYPDPlusLiquidMedium. 8.Shakeat30°Cfor90min. 9.Centrifugeathighspeedfor15sec. 10.Discardthesupernatantandresuspendin1mlofNaClSolution(0.9%)bygentlypipetting

upanddown. 11.Spread100µlofa1:10,1:100,and1:1,000dilutiononSD/–Leu,SD/–Trp,andSD/–Leu/–Trp

tocheckthetransformationefficiency,andonSD/–His/–Leu/–Trp+10mM3-ATtoselectforpositiveone-hybrids.

12.Incubatetheplatesat30°C(facedown)for2–7days,untilcoloniesappear. 13.CompareyourresultswiththoseshowninTableVIII.

TABLEVIII.CONTROLONE-HYBRIDCOTRANSFORMATIONS:EXPECTEDRESULTS

ControlStrain PlatedonSDMinimalMedia Phenotype(Growth)

PositiveControlY187[pGAD-Rec2-53+p53HIS2] –His/–Leu/–Trp+10mM3-AT +

NegativeControlY187[pGAD-Rec2-53+pHIS2.1] –His/–Leu/–Trp+10mM3-AT –

XI. Constructing&ScreeningaOne-HybridLibrarycontinued



TherearetwowaystoscreenaMatchmakertwo-hybridlibrary:

• Yeastmating

• Cotransformation

Themethodyouchoosedeterminestheprotocolyouwillnowusetoconstruct(andscreen)yourlibrary.Toscreenbyyeastmating,constructyourlibraryusingProtocolA.Toscreenbycotransformation,constructyourlibraryusingProtocolB.Foraquickcomparisonofthesetwoprotocols,referbacktoFigure7.

Important:Notethatyoumustpreparecompetentyeastcellsbeforestartinglibraryconstruction.PleasetakesometimetoreviewtheprocedureinAppendixB,andplanyourworkaccordingly.

ProtocolA:ScreenbyYeastMatingPLEASE READ ENTIRE PRoToCoL BEfoRE STARTING. Beforestarting: • PourSDagarplates.Youwillneed: SD/–Leu ~200 150-mmplates SD/–Leu ~5–10 100-mmplates SD/–Trp(seenotebelow)* ~5–10 100-mmplates SD/–Leu/–Trp ~5–10 100-mmplates SD/–His/–Leu/–Trp(seenotebelow)*~50 150-mmplates SD/–Ade/–His/–Leu/–Trp/X-α-Gal ~50 150-mmplates AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hr

beforeplatinganytransformationmixtures. * Note:AsexplainedintheListofAdditionalMaterialsRequired(SectionIII),thedropoutsupplementsrequired

tomakethesemediaarenotsupplied.YoumustobtainthesesupplementsseparatelyfromacommercialsupplierorpreparethemyourselfusingtherecipeinAppendixCoftheYeastProtocolsHandbook(PT3024-1).

• Plancontrols(Steps7–8)Controlsshouldbedoneinparallelwithexperimentalwork. • PrepareFreezingMedium:YPDmediumwith25%(v/v)glycerol. • PreparePEG/LiAcSolution(SectionIII).

1.TransformyeaststrainAH109withdscDNAandpGADT7-Rec. a. Preparecompetentyeastcells(AppendixB). b. Inasterile,prechilled,15-mltubecombinethefollowing: • 20µldscDNA(fromSectionIX.I,Step16) • 6µlpGADT7-Rec(0.5µg/µl) • 20µlHerringTestesCarrierDNA,denatured*

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.

c. Add600µlofcompetentcellstotheDNA. d. Gentlymixbyvortexing. e. Add2.5mlPEG/LiAcSolution. f. Gentlymixbyvortexing. g. Incubateat30°Cfor45min.Mixcellsevery15min. h. Add160µlDMSO,mix,andthenplacethetubeina42°Cwaterbathfor20min.Mix

cellsevery10min. i. Centrifugeat700xgfor5min. j. Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium. Note:YPDPlusisspeciallyformulatedtopromotetransformation.DonotusestandardYPDmediumfor

thisstep.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolA



k. Incubateat30°Cwithshakingfor90min. l. Centrifugeat700xgfor5min. m. Discardthesupernatantandresuspendin30mlofNaClSolution(0.9%).

2.SelecttransformantsonSD/–Leuplates. a. Spread150µloneach150-mmplate(~200platestotal). Note:Tocheckthetransformationefficiency,spread100µlofa1:10,1:100,1:1,000,and1:10,000dilution

on100-mmSD/–Leuplates.

b. Incubateplatesupsidedownat30°Cuntilcoloniesappear(~3–6days). c. Calculatethetransformationefficiency. Expectedresults:≥1x106transformants/3µgpGADT7-Rec 3.Harvest(pool)transformants. a. Chillplatesat4°Cfor3–4hr. b. Add5mlFreezingMediumtoeachplate. c. Usesterileglassbeadsandgentleswirlingtodislodgethecellsintotheliquid. d. Combineallliquidsinasterileflask.Mixwell. e. Checkthecelldensityusingahemacytometer.Ifthecelldensity≤2x107cells/ml,

reducethevolumeofthesuspensionbycentrifuging. f. Aliquot(1-ml)andstoreat–80°C(notlongerthan1year). g. Todeterminethelibrarytiter,spread100µlofa1:100,1:1,000,and1:10,000dilution

on100-mmSD/–Leuplates.Incubateat30°Cuntilcoloniesappear(~2–3days).Countthecolonies(cfu)andcalculatethenumberofclonesinyourlibrary.

4.Matethelibraryhoststrainwithyourbaitstrain. a. Thawa1-mlaliquot(≥2x107cells)ofyourAH109[library]inaroomtemperature

waterbath. b. Combinethe5-mlY187[bait]culturefromSectionVIII.C.7(≥1x109cells/ml)andthe

1-mlaliquotofAH109[library]cells(≥2x107cells/ml)inasterile2-Lflask. Note:Theflasksizemustbeatleast2Ltopermitsufficientaerationofthematingcultureatlow-speedswirling.

c. Add45ml2XYPDA/Kan(50µg/ml).Swirlgently. d. Rinsecellsfromlibraryvialwithtwo1-mlaliquotsof2XYPDA/Kan(50µg/ml). e. Incubateat30°Cfor20–24hrwithgentleswirling(30–50rpm). Note:Low-speedswirlingisnecessarytokeepcellsfromsettlingtothebottomoftheflask.However,

shakingthecultureatspeeds>50rpmwillsignificantlyreducematingefficiency.

f. After20hrofmating,checkadropofthematingcultureunderaphase-contrastmicroscope(400X).Ifzygotesarepresent,allowmatingtocontinueforfourmorehours.Otherwise,continuetoStepg.

Note:Azygotetypicallyhasathree-lobedshape,thelobesrepresentingthetwohaploid(parental)cellsandthebuddingdiploidcell.

g. Transferthematingmixturetoasterile100-mlcentrifugebottle.Centrifugeat1,000x g for 10 min. Meanwhile, rinse the mating flask twice (50 ml each rinse) with0.5XYPDA/Kan(50µg/ml).Combinetherinsesandusethemtoresuspendthepellet.

h. Centrifugeat1,000xgfor10min.Resuspendthecellpelletin10mlof0.5XYPDA/Kan(50µg/ml).Measurethetotalvolumeofcells+medium.

5.Selectforyeastdiploidsexpressinginteractingproteins a. Todeterminethematingefficiency,spread100µlofa1:10,000,1:1,000,1:100,and

1:10dilutionofthematingmixtureonthreemedia(100-mmplates): •SD/–Leu •SD/–Trp •SD/–Leu/–Trp b. SpreadremainingmatingmixtureonTDOorQDOplates(200µlcells/150-mmplate). •TDOstandsforTripleDropoutMedium:SD/–His/–Leu/–Trp •QDOstandsforQuadrupleDropoutMedium:SD/–Ade/–His/–Leu/–Trp SeeFigure11.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolAcontinued



c. Incubateat30°Cuntilcoloniesappear. After2–3days,somecolonieswillbevisible,butplatesshouldbeincubatedforatleast5

daystoallowslowergrowingcolonies(i.e.,weakpositives)toappear.Ignorethesmall,palecoloniesthatmayappearafter2daysbutnevergrowto>1mmindiameter.TrueAde+,His+coloniesarerobustandcangrowto>2mm.Also,Ade+coloniesarewhiteorlightpink,whereasAde–colonieswillslowlyturnredonadenine-limitedmedium.

d. ScoreforgrowthonSD/–Leu,SD/–Trp,andSD/–Leu/–Trp.CalculateMatingEfficiencyandNumberofColoniesScreened(Part6).

e. ForcoloniesgrowingonTDOmedium:ReplicaplatecoloniesontoQDOmedium.Incubateat30°Cfor3–8days.

f. ChooseAde+,His+coloniesforfurtheranalysis. Not all of the colonies surviving this selection will be true two-hybrid positives.

However,themostcommonclassoffalsepositiveswillbeeliminatedbyscreeningforexpressionofADE2 and HIS3(Jameset al.,1996).OthertypesoffalsepositivescanbeeliminatedasdescribedinSectionXIII.

g. StreakoutAde+/His+coloniesonfreshSD/–Ade/–His/–Leu/–Trp/X-α-Galmasterplatesandgrowfor2–4daysat30°C.HavingX-α-GalpresentinthemediumenablesyoutotestAde+/His+coloniesfortheactivationofathirdreporter:MEL1,which encodesα-galactosidase,asecretedenzymethathydrolyzesX-α-Galtoproduceablueendproduct.

h.SealthemasterplateswithParafilmandstoreat4°C.Ifdesired,prepareglycerolstockculturesofinterestingclonesandfreezeat–70°Cforlong-termstorage.

6.CalculateMatingEfficiency&NumberofClonesScreened a.Countthecolonies(cfu)growingontheSD/–Leu,SD/–Trp,andSD/–Leu/–Trpdilution

platesthathave30–300cfu. b. Calculatetheviablecfu/mloneachtypeofSDmedium: cfu =#viablecfu/ml Vol.plated(ml)xdilutionfactor #cfu/mlonSD/–Leu=viabilityofY187partner #cfu/mlonSD/–Trp=viabilityofAH109partner #cfu/mlonSD/–Leu/–Trp=viabilityofdiploids

Colony growth indicates an interaction between the two-hybrid proteins

Mate

DNA-BD/baitMarker: TRP1

AD/fusion libraryMarker: LEU2

Plate culture on SD/–His/–Leu/–Trp

Plate culture onSD/–Ade/–His/–Leu/–Trp

Medium-stringency

Replica plate to SD/–Ade/–His/–Leu/–TrpHigh-stringency

Y1�7 AH109

Figure11.ScreeninganADfusionlibraryfortwo-hybridinteractions.Usethestringencyofyourchoicetoscreenforinteractingproteins.Note:highstringencyselectionsresultinfewercolonies,andreducethenumberoffalsepositives.However,weakinteractionsmaybemissed.




c. Compare theviablecfu/mlof the twomatingpartners.Thestrainwith the lowerviabilityisthe“limiting”partner.Inthislibraryscreeningprotocol,theAH109[library]strainshouldbethelimitingpartnertoensurethatthemaximumnumberoflibrarycellsfindamatingpartner.Inacontrolcross,eitherstraincouldbelimiting.

d. Calculatethematingefficiency(i.e.,%Diploid): #cfu/mlofdiploids x100=%Diploid #cfu/mloflimitingpartner Note:Ifthematingefficiencywas



g. Removesupernatantandresuspendin1mlofYPDPlusLiquidMedium. h. Incubateat30°Cfor90min. i. Centrifugeathighspeedfor15sec. j. Discardthesupernatantandresuspendin1mlofNaClSolutionbygentlypipetting

upanddown. k. Spread100µlofa1:10,1:100,and1:1,000dilutiononto100-mmplatescontaining

SD/–LeuorSD/–Trp,dependingonthenutritionalmarkercarriedbytheplasmid(TableIX).

l. Incubatetheplatesat30°C(facedown)for2–4days,untilcoloniesappear. m. Pickthelargestcoloniesandrestreakthemonthesameselectionmedium.Seal

thesemasterplateswithParafilmandstoreat4°C(notlongerthan1month).UsethesecoloniesforthecontrolmatingsinSection8.

8.ControlsforProtocolA:Small-ScaleYeastMating

a. Pick one colony of each type—i.e.,AH109[pGBKT7-RecT],Y187[pGBKT7-53], andY187[pGBKT7-Lam]—touseinthemating.Useonlylarge(2–3-mm),fresh(2weeks),prepareglycerolstockculturesandfreezeat–70°C.ThesediploidsareusefulasreferencestrainswhenyouwishtocheckanewbatchofSDselectionmedium.

TABLEX.SET-UPFORCONTROLTwO-HYBRIDMATINGS

Cross PlateonSDMinimalMediaa Phenotype (100-mmplates) Mel1 His/Ade

PositiveControlAH109[pGADT7-RecT]xY187[pGBKT7-53] –Leub –Trpb –Leu/–Trpb –Ade/–His/–Leu/–Trp/X-α-Gal Blue +

NegativeControlAH109[pGADT7-RecT]xY187[pGBKT7-Lam] –Leub –Trpb –Leu/–Trpb

–Ade/–His/–Leu/–Trp/X-α-Gal nocolonies –

a Spread100-µlaliquotsof1:10and1:100dilutionsofthematingculture.bUsetheseplatestocalculatethematingefficiency.




ProtocolB:ScreenbyCotransformationPLEASE READ ENTIRE PRoToCoL BEfoRE STARTING. Beforestarting: • PourSDagarplates. SD/–Leu ~5–10 100-mmplates SD/–Trp ~5–10 100-mmplates SD/–Leu/–Trp ~5–10 100-mmplates SD/–His/–Leu/–Trp ~50 150-mmplates SD/–Ade/–His/–Leu/–Trp/X−α-Gal ~50 150-mmplates AllowSDagarplatestodryatroomtemperaturefor2–3daysorat30°Cfor3hr

beforeplatinganytransformationmixtures. • Plancontrols(Step3)Controlsshouldbedoneinparallelwithexperimentalwork. • PrepareFreezingMedium:YPDmediumwith25%(v/v)glycerol. • PreparePEG/LiAcSolution(SectionIII).

1.CotransformyeaststrainAH109withdscDNA,pGADT7-Rec,andpGBKT7/bait. Important:Note thatyoumustpreparecompetentyeast cellsbeforestarting library

construction.PleasetakesometimetoreviewtheprocedureinAppendixB,andplanyourworkaccordingly.

a. Preparecompetentyeastcells(AppendixB). b. Inasterile,15-mltubecombinethefollowing: • 20µl dscDNA(fromSectionIX.I,Step16) • 6µl pGADT7-Rec(0.5µg/µl) • 5µg pGBKT7/baitplasmidDNA(≤10µl) • 20µlHerringTestesCarrierDNA,denatured*

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe15-mlreactiontube.

c. Add600µlofcompetentcellstotheDNA. d. Gentlymixbyvortexing. e. Add2.5mlPEG/LiAcSolution. f. Gentlymixbyvortexing. g. Incubateat30°Cfor45min.Mixcellsevery15min. h. Add160µlDMSO,mix,andthenplacethetubeina42°Cwaterbathfor20min.Mix

cellsevery10min. i. Centrifugeat700xgfor5min. j. Discardthesupernatantandresuspendin3mlofYPDPlusLiquidMedium. Note:YPDPlusisspeciallyformulatedtopromotetransformation.DonotusestandardYPDmediumfor

thisstep.

k. Incubateat30°Cwithshakingfor90min. l. Centrifugeat700xgfor5min. m. Discardthesupernatantandresuspendin6mlofNaClSolution(0.9%).

2.Selectfortransformantsexpressinginteractingproteins a. SpreadthecotransformationmixtureonTDOorQDOplates(150µlcells/150-mm

plate). •TDOstandsforTripleDropoutMedium:SD/–His/–Leu/–Trp •QDOstandsforQuadrupleDropoutMedium:SD/–Ade/–His/–Leu/–Trp SeeFigure11. Note:Determinethetransformationefficiencyasfollows: i. Removea30-µlaliquotfromthe6-mlsuspensionanddilutewith720µlofNaClSolution(forafinal

volumeof750µl).

ii. Spread150-µlaliquotsofthisdilutionontwoseparate150-mmplates:SD/–Leu/–TrpandSD/–Leu.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolB



b. Incubateat30°Cuntilcoloniesappear. After2–3days,somecolonieswillbevisible,butplatesshouldbeincubatedforatleast

5daystoallowslowergrowingcoloniestoappear.Ignorethesmall,palecoloniesthatmayappearafter2daysbutnevergrowto>1mmindiameter.TrueAde+,His+coloniesarerobustandcangrowto>2mm.Also,Ade+coloniesarewhiteorlightpink,whereasAde–colonieswillslowlyturnredonadenine-limitedmedium.

c. ScoreforgrowthontheSD/–LeuandSD/–Leu/–Trpdilutionplates.

i.CountthecoloniesgrowingonSD/–Leu. #coloniesx1,000=#transformants/3µgpGADT7-Rec. Expectedresults:≥1x106transformants/3µgpGADT7-Rec ii.CountthecoloniesgrowingonSD/–Leu/–Trp. #coloniesx1,000=#clonesscreened Expectedresults:≥5x105clones/library d. ForcoloniesgrowingonTDOmedium:ReplicaplatecoloniesontoQDOmedium.

Incubateat30°Cfor3–8days. e. ChooseAde+/His+coloniesforfurtheranalysis. Not all of the colonies surviving this selection will be true two-hybrid positives.

However,themostcommonclassoffalsepositiveswillbeeliminatedbyscreeningforexpressionofADE2 and HIS3 (James et al.,1996).OthertypesoffalsepositivescanbeeliminatedasdescribedinSectionXIII.

f. StreakoutAde+/His+coloniesonfreshSD/–Ade/–His/–Leu/–Trp/X−α-Galmasterplatesandgrowfor2–4daysat30°C.HavingX-α-GalpresentinthemediumenablesyoutotestAde+/His+coloniesfortheactivationofathirdreporter:MEL1,which encodesα-galactosidase,asecretedenzymethathydrolyzesX-α-Galtoproduceablueendproduct.

3.ControlsforProtocolB:Small-ScaleYeastCotransformation Usethissmall-scalecotransformationprotocoltoproducethefollowingtwocontrolstrains.

• SV40 Large T PCR Fragment • pGADT7-Rec• pGBKT7-53

Recombination

in vivoAH109[pGADT7-RecT + pGBKT7-53]

Two-Hybrid Cotransformation Controls

Control Strain[plasmids]Cotransform AH109 with:

1. Positive Control

• SV40 Large T PCR Fragment • pGADT7-Rec• pGBKT7-Lam

Recombination

in vivoAH109[pGADT7-RecT + pGBKT7-Lam]�. Negative Control

TABLEXI.SET-UPFORCONTROLTwO-HYBRIDCOTRANSFORMATIONS

Component #1PositiveControl(µl)#2NegativeControl(µl)

SV40LargeTPCRFragment(25ng/µl 5.0 5.0pGADT7-Rec(SmaI-linearized;500ng/µl) 0.5 0.5pGBKT7-53(500ng/µl) 0.5 —pGBKT7-Lam(500ng/µl) — 0.5HerringTestesCarrierDNA(10mg/ml),denatured* 5 5AH109CompetentYeastCells 50 50PEG/LiAcSolution 500 500

*Transfer~50µlofHerringDNAtoamicrocentrifugetubeandheatat100°Cfor5min.Then,immediatelychilltheDNAbyplacingthetubeinanicebath.RepeatoncemorebeforeaddingtheDNAtothe1.5-mlreactiontube.

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolBcontinued



a. Preparecompetentyeastcells(AppendixB). b. Setuptwo1.5-mlmicrocentrifugetubes.AddDNA,competentyeastcells,andPEG/

LiAcSolutionusingthevolumesandintheorderindicated(TableXI). c. Mixthoroughlybygentlyvortexing. d. Incubateinawaterbathat30°Cfor30min.Vortexgentlyevery15min. e. Add20µlofDMSOtoeachtube,mix,andthenplacethetubeina42°Cwaterbathfor

20min.Vortexgentlyevery5min. f. Centrifugeathighspeedfor15sec. g. Removesupernatantandresuspendin1mlofYPDPlusLiquidMedium. h. Incubateinawaterbathat30°Cfor90min.Mixevery15minbygentlyvortexing. i. Centrifugeathighspeedfor15sec. j. Discardthesupernatantandresuspendin1mlofNaClSolutionbygentlypipetting

upanddown. k. Spread100µlofa1:10,1:100,and1:1,000dilutiononto100-mmSDagarplates:

• SD/–Leu/–Trp Tocheckthecotransformationefficiency

• SD/–Ade/–His/–Leu/–Trp/X−α-Gal Toselectforcotransformantsexpressinginteractingproteins

l. Incubatetheplatesat30°C(facedown)for2–4days,untilcoloniesappear. m. CompareyourresultswiththoseshowninTableXII. n. Pickthelargestcoloniesandrestreakthemonthesameselectionmedium. o. Aftercolonieshavegrown,sealthesemasterplateswithParafilmandstoreat4°C.

Forlongtermstorage(>2weeks),prepareglycerolstockculturesandfreezeat–70°C.ThesestrainsareusefulreferencesforcheckingnewbatchesofSDselectionmedium.

TABLEXII.CONTROLTwO-HYBRIDCOTRANSFORMATIONS:EXPECTEDRESULTS

ControlStrain PlateonSDMinimalMedia Phenotype Mel1 His/Ade

PositiveControlAH109[pGADT7-RecT+pGBKT7-53] –Ade/–His/–Leu/–Trp/X-α-Gal Blue +

NegativeControl

AH109[pGADT7-RecT+pGBKT7-Lam] –Ade/–His/–Leu/–Trp/X-α-Gal nocolonies –

XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolBcontinued



This section presents strategies for verifying and analyzing protein-protein and protein-DNAinteractions.Figure12providesadetailedoverview.

A. RetestthePhenotype 1.Library transformants may contain more than one AD/library plasmid, which can

complicatetheanalysisofputativepositiveclones.Thus,itisagoodideatorestreakthepositivecoloniesonSDdropoutplates2–3timestosegregatetheAD/libraryplasmids.YoushouldrestreakonanSDdropoutmediumthatselectsforboththelibraryandbaitplasmidsaswellasfortheone-hybridortwo-hybridinteraction:

a. Restreakpositiveone-hybridclonesonSD/–His/–Leu/–Trp+optimal[3-AT]. Restreakpositivetwo-hybridclonesoneitherTDOorQDOmedium.Keepinmind

thatQDOisamorestringentselection.Reminder: TDOstandsfortripledropoutmedium=SD/–Ade/–Leu/–TrporSD/–His/–Leu/–Trp QDOstandsforquadrupledropoutmedium=SD/–Ade/–His/–Leu/–Trp b. Incubateplatesat30°Cfor4–6days. 2.[Optional]Testthephenotypefurtherbyassayingforathirdreportergeneorbyselecting

fortheinteractionunderdifferentconcentrationsof3-AT. • Two-hybridcolonies:Replicaplateortransferwell-isolatedcoloniestoSD/–Ade/–His/–

Leu/–TrpplatescontainingX-α-Gal

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