Southern Blot Protocol

Day11.DigestDNAfor6hours(orovernight)BSA10mg/ml0.522.65ulof10ugGenomicDNARnaseA10mg/ml0.1inTEmixedto7.35ulcocktailSpermidine100nM0.75persample10Xenzymebuffer3.0enzyme3.02.Pouralarge1%gel(5gramsagarosein500ulTAE,10-20ul10mg/mlEtBr/500mlofgel),coverandplaceinfridgetillreadytouse

Day21mix3ulBlueJuicepersampleandloadintolane2add2ulbluejuicetoMarkermix(Add.5ugunlabeledlamBDaHindIIIto~3500counts(~.5ul)of32Plabeledlambda,heatlambdato56degreesfor3minutes,chillonice1minute,centrifuge3rungelat400–500mAfor3–4hoursoruntildyehasreachednextcomb4takeapictureofthegel(withruler)5soakgelinSoln1onshakerfor15min,repeatwithnewsoln1(denaturingtheDNA)6soakgelinsoln2onshakerfor30min,repeatwithnewsolnfor20minutes7cut2piecesofblottingpaperand1pieceofnitrocellulosetosizeofthegel8gentlyplacenitrocelluloseontopofalayerofddH2Oandletthewatersoakinonitsown9oncecompletelysoaked,drainH2Oandletpapersoakintransferbuffer(soln2)***DONOTLETNITROCELLULOSEDRYOUT***10PlaceaPlexiglasplateoveraglassdishandpoursoln2intothedish11CutapieceoffilterpaperlargeenoughtolayacrossthePlexiglasandhavetheendssoakinginthebuffer12SoakfilterpaperinthebufferandthenlayacrossthePlexiglas,centerfirst,sothatthereareNOBUBBLES(**UseaplasticPipettetorolloutbubbles**)13PlacegelINVERTEDontofilterpaper->NOBUBBLES14Removenitrocellulosepaperfrombufferandlayontothegel->NOBUBBLES15Soakapieceofblottingpaperinbufferandlayitontop->NOBUBBLES16Placeasecond,dry,pieceofblottingpaperontop17Placesaranwraponallsidesofthegelsothatallsolnmustpassthroughthemembraneandnottravelaroundit18Carefullyplaceastackofbrownpapertowels3-4incheshighontopoftheblottingpaper19PlaceapieceofPlexiglasontopofthepapertowels20Centerafull1-2literbottleontopofthestack21Blotovernight

Day31Carefullyremovethestackofpapertowelsandblottingpaper2Removethenitrocellulose,flipitoverandwriteanLontheupperleftcornerwithaballpointpen(fororientation)3Placeitonfilterpaper,viewitunderUVlight,andmarkthelanes4Tapelightlyb/w2piecesoffilterpaper5Bake@80degreesinvacuumovenfor2hours6SoakinddH₂O1-2min7PreparePrehybridizationandHybridizationsolnandwarmat42degrees(add55ul10%SDSto11mlPrehybridizationsoln)(add55ul10%SDSto11mlHybridizationsoln)8Placenitrocelluloseintohybridizationtube(DNAsideup,notfacingglass),smoothpapertowallstoensurethatitrotateswiththetubeanddoesnotcomefreeofthewall9Addprehybridizationsoln,puttubeintothehybridizationovenfor1–2hoursat42degrees(**Prepareprobeduringthistime**)10Denaturetheprobe->add1ulof1MNaOHforevery9ulofprobeandputinto37degreeH₂Obathfor10minutes11Pourouttheprehydridizationsoln12Mixtheprobeintothehybridizationsolninthebottomofthehybridizationtubeandthenmixoverthepaper13Hybridizeovernightat42degrees

Day41warm2containersofLowStringencybufferto65degreesinshaker2removeblotsanddumphybsoln/probeintoproperrADIoactivewastecontainer3soakblotinonecontainerofLSbufferfor15minutes4dumpandrepeatwashinLSbuffer2moretimes5doaforthwashinHSbufferasneeded.6Oncereadingisroughly.02-.04usingtheGeigercounteronthelowestsetting,placeblotbetweenfilterpapertoremoveexcessbuffer7tapeblottofilterpaper8wrapblotandpapercompletelyinsaranwrapandtapesealedatbottomoronback9putblotandfilmincartridgeindarkroom10expose2hoursin–80freezeranddevelopfilm11exposeovernightasneeded