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Matchmaker™ Library Construction & Screening Kits User Manual
PT3955-1 (PR742237)Published 20 April 2007
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Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 � Version No. PR74��37
Matchmaker™ Library Construction & Screening Kits User Manual
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
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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
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Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3955-1 4 Version No. PR74��37
Matchmaker™ Library Construction & Screening Kits User Manual
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
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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.
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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.
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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
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Matchmaker™ Library Construction & Screening Kits User Manual
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
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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
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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
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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
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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(
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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(
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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
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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
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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
ds cDNA with SMART™ III & CDS III anchors
GGG5'CCC
5'
GGG5' CCC
VI.ProtocolOverview:One-HybridLibraryConstruction&Screening
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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
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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
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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
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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
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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
ds cDNA with SMART™ III & CDS III anchors
GGG5'CCC
5'
GGG5' CCC
Figure8.Synthesisofhigh-qualitydscDNAusingSMARTtechnology.
IX.GeneratingacDNALibrary
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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
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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.
IX.GeneratingacDNALibrarycontinued
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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
IX.GeneratingacDNALibrarycontinued
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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.
IX.GeneratingacDNALibrarycontinued
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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.
IX.GeneratingacDNALibrarycontinued
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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
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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
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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 –
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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
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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.
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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.
XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolAcontinued
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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
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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.
XII. Constructing&ScreeningaTwo-HybridLibrary:ProtocolAcontinued
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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
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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
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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
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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