MakinglibraryDNAfromtheDNAwesendyou

ThelibraryisdistributedasindividualpoolsintheformofDNA.Youwillbesentaboutamicrogramofeachpoolavailable.TransformasuitableamountintoE.coli(anystrainsuitableformakingplasmidpreps).Selecttransformantswith40ug/mlkanamycinand/or50ug/mlampicillin.Obtain50,000coloniesforeachpool.ElutecoloniesfromplatesinLB;makea-70Cstockofthiseluate.DiluteeluateintoLBplusantibiotictogiveaculturewithanalmostsaturateddensity.Growat37Cforafewhours.MakeminiprepormidiprepDNA.

TransformingyeastwithDNAfromtheinsertionlibrary

OVERVIEW:MutagenizedDNAfromthelibraryisexcisedfromthebacterialvector.Itisthentransformedintoaleu2strainofyeast.Thisprocedureisoutlinedinthisfigure.Useofacircle-zerostrainwillpreventrecoveryofinsertionsinthe2-micronplasmid.Thebeststrategyistoscreenafewthousandtransformantsfromeachpool.Screening30,000transformantsshouldgiveyou95%coverageoftheyeastgenome.

Tominimizedoubleintegrants,transformationsshouldcontainthelowestamountofDNApracticable.Wethereforerecommendthatapilotexperimentbeperformedtodeterminetransformationefficiencyofthestrain,andconditionsthenbescaledupasappropriate.ThepilotprotocolgivenbelowusesamodifiedversionofthemethodofChenetal.(1992).Youshouldusewhatevertransformationprotocolworksbestinyourhands.

1. PlasmidDNAfrompoolsofthemTn3-mutagenizedgenomiclibraryisdigestedwithNotI.A2.1-kbbandfromthevectorshouldbeapparent,togetherwitharangeofbandsinthe8-kbregion.
2. A10-mlcultureoftheyeasthoststrainisgrowntoadensityof10⁷cells/ml(O.D.600of1).Useofsuchlogarithmically-dividingculturesincreasestransformationefficiency.
3. Cellsarepelletedandwashedoncewith5volumesofOneStepbuffer(0.2MLiAc,40%PEG4000,100mMbeta-mercaptoethanol).Thiswashisespeciallyimportantwhenculturevolumesareincreased.
4. CellsareresUSPendedin1mlofOneStepbuffercontaining1mgofdenaturedsalmonspermDNA.100ulaliquotsofthissuspensionarethenaddedtotubescontainingfrom0.1to1ugofNotIdigestedplasmidDNA.
5. Tubesarevortexedtomixthecontentsthoroughly,thenincubatedat45^ofor30minutes.
6. Cellsarepelletedandresuspendedin400ulofSC-leu.200ulisplatedontoSC-leumedium.Platesareincubatedat30^oCfor3to4days.

ScreeningforgeneexpressionusinglacZfusions

OVERVIEW:Transformantstrainscarryingin-framefusionsbetweenyeastgenesandlacZareidentifiedbyacolorassayforbeta-galactosidaseactivity.

• TomaximizedetectionoflacZfusionsexpressedatalowlevel,transformantcoloniesarepatchedtoYPADplatesatadensityof100perplate.
• Toidentifyvegetativelyexpressedgenes,cellsarereplicaplatedtoanSC-leuplateonwhichasterilediscofWhatman1Afilterpaperhasbeenplaced,andgrownovernightat30^oC.Othermediaorgrowthconditionscanbesubstitutedasdesired.Forade2strains,anytestmediashouldcontain80mg/lofadenine.
• Filtersareliftedfromtheplatesandplacedinthelidofa9-cmglasspetridish.Thislidisthenplacedinsideaclosed15-cmglasspetridishcontainingchloroformfor10to30minutes.Theminimumexposuretimenecessaryforaparticularyeaststraincanbedeterminedempirically.
• Filtersareplacedcolony-sideupontoX-Galplates(120ug/ml5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,0.1MNaPO4[pH7]and1mMMgSO4in1.6%agar)andincubatedat30^oCforupto2days.Theseplatescanbeverythin;theiruseincreasesthesignaloverthatobtainedbysimplysoakingthefiltersinabufferedX-galsolution.
• TransformantscarryingproductivelacZfusionsarerecoveredfromtheregrownYPADplates.ItisadvisabletosubsequentlymaintainselectionforLEU2whereverpossIBLe,assomemutationsaredeleteriousevenintheheterozygousstate.

Identificationofthegenomicsiteoftransposoninsertion

OVERVIEW:Todeterminethesiteoftransposoninsertion,genomicDNAimediatelyadjacenttothelacZsequencesisrescuedinEscherischiacoli.Tointroduceanoriginofreplication(ori),aplasmidmarkedwithURA3(pRSQ2-URA3,U64694)replacespartofthetransposonbyrecombinationbetweenplasmid-andtransposon-bornecopiesoflacZsequences.Thisprocedureisoulinedinthisfigure.YeastDNAisrecoveredfromthesetransformantsandcutwitha"recovery"enzyme(EcoRI,HindIIIEcoRV,PstI,ClaI,SalI,XhoI,KpnI).Thisreleasesasalinearsegmentthebacterialoriginofreplication,thebeta-lactamasegeneandaportionofthelacZgenewithadjacentyeastDNA;thisfragmentisthencircularizedandrecoveredinbacteria.pRSQ2ishighcopynumberinE.coli.Plasmidsaresequencedusingaprimercomplementarytothe5"endofthetransposon.Thisprocessrequiresthethreeprotocolsgivenbelow.

Alternativemethod:C.Friddle(http://genome-www.stanford.edu/group/botlab)hasdevelopedavectorettePCRrescueprotocolforlacZ-basedtransposons.

CAUTION!Twoormoreinsertioneventsmayhaveoccurredinupto10%ofthepopulation.Thesecanbeidentifiedbyexaminationofsegregationofthetransposon-borneLEU2Markerupontetraddissection.YoushouldbesurethatthestrainhasonlyonetransposonbeforeproceedingtorecoveringaplasmidcontaininggenomicDNA.Ifyouhaveusedthelibraryformutagenesis,youarestronglyadvisedtomakesurethatyourphenotypeislinkedtothetransposoninsertion,sincespontaneousmutationscanariseatothersites.Youcanwastetimerecoveringjunkifyoudon"tcheck.

Transformationofyeaststrains

Wesuggesttransformingtheyeastwith1-5ugofBamHI-digestedpRSQ2-URA3plasmidDNA,selectingthetransformantsonSC-leu-ura.Thisisatargettedreplacement,soefficiencywilldependonyourstrain.ThemethodofChenetal.(1992)givenabovecanbeused.Ifanamp^Rplasmidispresentintheyeaststraintobetransformed,adifferentmarkercouldbeclonedintothepRSQ2polylinkertoenableitsrecovery.

RecoveryofgenomicDNAfromyeaststrains

ForadetaileddiscussionofgenomicDNApreparationfromyeast,seePhilippsenetal.(1991).Hereisthemethodthatweuse.

1. Yeaststrainsaregrowntosaturationat30^oCin2mlofYPAD.Wesuggestusingacoupleoftransformantsforeachstrain.
2. Cellsarerecoveredbycentrifugationat13,000r.p.m.for1minute.Thesupernatantisremovedbyaspirationandcellsareresupendedin250ulof0.1MEDTA(pH7.5),14mMbeta-mercaptoethanolcontaining150ug/mlzymolyase.Cellsareincubatedat37^oCuntilspheroplasted.Overspheroplastingdoesnotaffectrecovery.
3. 50ulofminiprepmix(0.25MEDTA(pH8.5),0.5MTrisbase,2.5%SDS)isaddedtoeachtube.Samplesaremixedbyinversion,thenincubatedinawaterbathat65^oCfor30minutes.
4. 63ulof5MKAcisaddedtoeachsample.Samplesaremixedbyinversionandincubatedonicefor30minutes.
5. Samplesarespunat13000r.p.m.inamicrofugefor10minutes.Supernatantsaretransferredbypouringeachsampleintoanewtubecontaining720ulof100%ethanol.ADNAprecipitateshouldbevisible.Samplesaremixedbyinversionandspunfor5minutesasabove.
6. Tubesaredrainedthoroughly,and130ulTEcontaining1mg/mlRNAaseAisaddedtotheundriedpellets.ResupensionofDNAisgradualandoccursduringsubsequentincubationat37^oCfor35minuteswithoccasionalvortexing,DNAisreprecipitatedbyadditionof130ulofisopropanol.Samplesaremixedbyinversionandspunfor5minutesasabove.
7. Tubesaredrained.A70%ethanolwashmaybeperformedtoremovesalt.Finally,pelletsareairdriedandresupendedin40ulofTEwithincubationat37^oC.About10ugofgenomicDNAisobtained.

Plasmidrescue

1. 5ugofyeastgenomicDNAisdigestedovernightat37^oCwith5unitsof"recovery"enzyme(EcoRI,HindIII,SalI,EcoRV,PstI,ClaI,XhoI,orKpnI)inatotalvolumeof40ul.
2. 20ulofthesampleisrunonageltocheckdigestion.ThisgelmayalsobeusedforSouthernanalysis(seebelow).Theremainderisheatedto65^oCfor25minutestoinactivatetherestrictionenzyme,and215ulofH2O,25ulof10Xligasebufferand1ulofligase(400units)areadded.Tofavourintramolecularreactions,theDNAconcentrationintheligationshouldnotbeover10ug/ml,andcanbeaslowas2ug/ml.
3. Afterligationat16^oCfor4to16hours,DNAisprecipitatedbyadditionof125ulof7.5MNH4Acand375ulofisopropanolandrecoveredbycentrifugationat2190g(ormore)for20minutes.
4. TheDNApelletiswashedoncewith70%ethanol,thenresuspendedin6-20ulofTE.3ulofthisistransformedintoE.coli,(weuseelectroporation)selectingforampicillinresistance.Dominiprepsofseveralcoloniesforeachstrain.
5. Rescuedplasmidscanbeanalyzedbydouble-digestionwithBamHIandthe"recovery"enzyme.Desiredplasmidsdisplaya2.85-kbbandcontainingvectorsequences(seeFigure1b)plusadditionalband(s)fromgenomicDNA.Ifyouget"mystery"plasmids,tryadifferenttransformant/recoveryenzyme.
6. DNApreparationsmaybesequencedusingaprimerfromlacZsequencesinsidetheterminalrepeat,egthe-40primer(#1212,NewEnglandBiolabs).Onlytrustyoursequenceuptothefirstsitefortherecoveryenzyme,asotherfragmentscangetclonedinduringcircularization.

SequenceoflacZendofmTn3-lacZ/LEU2:

Bases1-38aretheterminalrepeat,alsopresentonthevector.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGggggATCCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGCGTTACCCAACTTAATCG

TheaccessionnumberformTn-lacZ/LEU2isU35112.

TheaccessionnumberforpRSQ2-URA3isU64694.

Alternativerescuestrategies

pRSQ

• DiagramoutlininguseofpRSQ
• U34887,GenbankentryforpRSQ

YIp5

• DiagramoutlininguseofYIp5
• L09157,GenbankentryforYIp5

VectorettePCR

CarlFriddlehasdevelopedavectorettePCRprotocolforidentifyingthesiteofinsertion.Thiscanbefoundathttp://genome-www.stanford.edu/group/botlab/protocols/vectorette.html.

Transferringthedisruptionalleletootherstrains:

WhenpRSQ2-URA3integratesintothetransposonitcreatesan11.7kbinsertion.Thiselementisnotcleavedbythefollowingenzymes:AvrII,BglII,BspEI,EagI,MscI,NaeI,NheI,NruI,NotI,PmlI,SmaI,SnABI,SpeI,SphI,XmaI.Theseenzymescanthereforebeusedtorecoveralargeplasmidcontainingsequencesboth5"and3"tothetransposoninsertion.Wehavesuccessfully"moved"disruptionsbythisstrategy.NotethatthisstrategydoesnotmovethealleleasalacZ-fusion.yIP5canbeusedifthisisrequired.

Antibioticsused: