AnalysisofESCellClonesbyMini-SouthernInitialCloning1.Aspirateselectivemediaofftheplatescontainingcoloniesofinterest.WashtheplatestwicewithPBS.Afteraspiratingthesecondwash,addenoughPBStocoverthesurfaceofthedish.2.Preparethe96-wellU-bottomcloningdishbypipetting25uloftrypsinintoeachwellusingamultichannelpipettor.Uptotwofullplatesofclonescanbepickedatonesittingwithoutproblemsofthetrypsindrying.3.Pickcoloniesfromthewashedplatesandtransferthemintothetrypsinsolution,onecolonyperwell.Proceeduntilcolonieshavebeenclonedintoeachofthe96wells.Approximatelyonehourwillhaveelapsedbytheendoftheprocess(assumingapickingspeedof100-150colonies/hour),enoughtimetoensurecompletetrypsinizationofthecolonies.Therefore,nofurtherincubationisnecessary.4.Usingthemultichannelpipettor,add25ulofM15mediaperwell.Pipetteup-and-downseveraltimestodisaggregatethecells.5.Transfertheclonestoanappropriatelylabelled96-wellfeederplateandallowthecoloniestogrowunderselection.Thecellswilltakeabout3-5daystogrowtosuchaconfluenceastoturnthemediayellowatone-dayintervals.DuplicatePreparation1.Oncetheclonesreachconfluence,trypsinize,duplicate,andfreezethecells[refertotheprotocol"FreezingEmbryonicStemES)CellClonesin96-WellPlates"].2.Allowthecellstogrowonthegelatinized96-wellduplicateplates.RefeedtheplateswithfreshM15mediaastheoldmediaturnsyellowuntilthecellsarereadyforuse.Mini-SouthernAnalysis1.Whenthecellsareready,washtheplatestwicewithPBSandaspirate.Usingthemultichannelpipettor,add50ulofLysisBuffer[10mMTrispH7.5,10mMEDTApH8.0,10mMNaCl,0.5%Sarcosyl,and1mg/mlProteinaseK(addedfresh)]perwell.Note:Fromthispointon,allmanipulationsaretobeperformedoutsidethetissueculturefacilities.2.Incubatetheplatesovernight@60oCinahumidifiedchamber(suchasaplasticcontainerwithawetspongeontheinside).3.Nextday,prepareafreshsolutionof75mMNaClinethanol(add150ulof5MNaClper10mlofcoldabsoluteethanolandmixwell;thesaltwillprecipitate,butthisisoflittleconsequence).4.Usingthemultichannelpipettor,add100uloftheNaCl/ethanolsolutionperwell.Allowtheplatetorestonthebench@roomtemperaturefor15-30minutes,oruntiltheprecipitatedDNAisclearlyvisIBLeunderlow-powermagnification.TheDNAadherestotheplastic,solookattheperimeterofeachwelltoseetheprecipitatedDNA.5.Inverttheplatetodiscardthesolution(theDNAwillremainadheredtotheplate).Usingthemultichannelpipettor,add70%ethanoltowasheachwell.Alternatively,asquirtbottlemaybeused,butastrongstreamcoulddetachtheDNAfromtheplate.Inverttheplatetodiscardthe70%ethanolandrepeatthewash2-3times.6.Afterthefinalwash,inverttheplate,discardthe70%ethanol,andallowtheplatetoair-dryafewminutes.7.Whiletheplateisdrying,preparetheRestrictionEnzymeCocktail(1XRestrictionBufferspecifiedfortheenzymebeingused,1mMSpermidine,100ug/mlBovineSerumAlbumin,and10-15unitsofenzyme).8.Usingthemultichannelpipettor,add30ulofRestrictionEnzymeCocktailtoeachwellandmixbypipettingup-and-down.Changetipsbetweenonerowandthenext.9.Oncethecocktailhasbeenaddedtoallthewells,incubatetheplatesovernight@37oCinahumidifiedchamber.10.Nextday,preparetheagarosegel(s)forelectrophoresis.Preparealargegeltray(15cmx24cm)withthree33-teethcombs(add3extrateethpercomb,securedwithtape)evenlydistributedalongthelengthofthetray.Pour300mlofmoltenagaroseintothetray;removeanybubbleswithaneedle.Allowthegeltosolidifyforabout30minutes.Thissizeofgel(3laneswith99wellstotal)willaccommodateone96-wellmini-Southerndigestplateat32samplesperlaneplusonewellperlaneformolecularweightMarkers.11.Removethe96-wellmini-Southerndigestplatefromtheincubatorandadd4-5ulofloADIngbuffertoeachwell.Loadthegel(30-35ulperwell)andrun@80Vforapproximately4hours.Whendecidinghowfartoallowthesamplestomigrate,takeintoaccountthesizeofthefragment(s)beingdistinguished.Thegelcanberunfurtheraslongasthebandsfromonelaneofsamplesbeingdetectedwithaprobedonotoverlapthebandsfromthenextlaneofsamples.12.Aftertheelectrophoresisiscomplete,transfertheDNAtoGeneScreenPlusmembranesaccordingtotheusualprotocol[refertotheprotocol"GenomicDNA:RestrictionEnzymeDigests,AgaroseGelElectrophoresis,andSouthernTransfer(Blotting)"].NOTES:Thisprotocolmaynotworkefficientlyforallrestrictionenzymes.Therefore,makeapilotexperimenttotestthedesiredenzyme(s)beforeproceedingtothelargeexperiment.BeforetransferringtheDNAtomembranes,discardtheareasoftheagarosegelthatarenotneededforprobing.FromtheLaboratoryofDr.AllanBradleyBaylorCollegeofMedicine,Houston,Texas