1.Runthegelasnormal.Oftenforgenomicsouthernsitisdesirabletorunlonggels(18cm)over4-6hrs.2.PhotographthegelwitharuleradjacenttothemolecularweightMarkersasareference.3.Alkalitransferbuffer=0.5MNaOH(20g/lt),1.5MNaCl(87.66g/lt).Prepare1litreforonegelandanother750mlforeachadditionalgel.BEWAREThisbufferisverydangerous,capableofcausingsevereeyedamage.Usethelargevolumesinvolvedinthisprocedurewithcareandwearprotectiveglasses.4.Addthegelto250mlalkalitransferbuffer,plus125mlforeachadditionalgel.5.Placegelonrockerwithbuffersolutionfor20mins.NOTElow%agarosegelsmustbeagitatedslowlytopreventtearing.6.Keepremaining500mlforthetransfertank.Wearglovesforthefollowingsteps7.Cut2piecesoflarge3MMpaper(wicks)and2piecesabout2mmsmallerthanthegeloneachedgeandonepieceofnylon(HybondN,Amersham)thesamesizeasthegel.Largegel(HFI)MediumgelMinigel2(14x19cm)2(15.2x10)2(9x5.2)2(14x32cm)2(15.2x32)2(18.5x5.2)Cutastackofpapertowelingabout2mmsmallerthanthegel.Thestackneedstobeabout6cmthick.8.PrewetnyloninDDW,thensoakinalkalitransferbuffer.9.Addbuffertotransfertanktotheleveloftheplatform.Wetthelongwicksintransferbufferandplaceinthetank.10.Placegelonplatformandspoonontransferbuffer.AddtheNylonandsmoothoutanybubbleswiththebackofyourfinger.11.Addthe2slightlysmaller3MMfilters,thefirstprewetted,theseconddry.12.Addthestackofpapertowels.NOTEitisveryimportantthattheedgesofthetoweldonottouchthewicksthatthegelissittingonorthetransferwillbe"shortcircuited".13.Topthestackwithaglassorplasticplateandaweight(abottlewithabout200-300mlH2Oisideal).Toomuchweightcompressesthegelandterminatesthetransferearly.14.Allowtotransfero/nandthenremovethestackcarefully.Markthepositionofthewellsonthefilterwithabiro(andnotepositionofwell#1)beforeremovingthefilter.Soakthefilterin0.5MTrispH7.5,1.5MNaClfor5min.afterremovalfromthegel.15.Addfilterto200ml2XSSCandallowtosoakwithoutagitationfor5".16.Removefilterandblotdryandbakeat80?for2hrorplaceinautoclavefor10"whennotoperatingbutwarm.17.Prehybridizewith10mlAqua.hyb.(Reagents),1%SDSand100ug/mlboiledherringspermDNAfor20-30minutes(clonedDNAsouthern)toseveralhrs(genomicsouthern).18.Boilprobefor3"andaddto3mloffreshhybsolution/SDS/HerringDNA.Squeezeprehybfromthebagandaddprobe.Seal,avoidingairbubblesanddistributetheprobewell.Incubatefor4-6hr(clonedDNA)to16-20hr(genomicsouthern).Washesareperformedin2to0.2XSSC,0.1%SDSdependingonhomologyofprobetotarget
