WormgenomicSouthernblots

I.PreparingwormgenomicDNA:requires1-2daystoseedagaroseplates,afewdaysforthewormstogrow,1-2daystopreptheDNA

1.SeedlargeagaroseplateswithHB101.Agaroseispreferredoveragarsincethelattercontainsmoreinhibitorsofrestrictionenzymes.HB101ishealthierthanOP50,andproducesathickerlawnallowingahigheryieldofworms.Expectayieldof~10-130µgofgenomicDNAperlargeplate,averagingaround40µg,soscaleupaccordingly.ForSoutherns,youwilluse1-5µgperlane.

2.Harvestthewormsbeforetheplatesstarve.Afterthebacteriaaregone,thewormsbeginbreakingdowntheagaroseandreleasingenzymeinhibitors.Itisbesttoharvestwhenthewormsareformingawaveacrosstheplateastheyeatthelastbacteria.

3.RinsethewormsofftheplateinM9,spindownbriefly(30")inaclinicalcentrifuge.MaywanttowashoncewithmoreM9toremovebacteria.

4.Optionalstep:thisisusuallytotallyunnecessary.Toremoveagarosecontamination,canfloatthewormsinsucrose.ResUSPendthewormsin5mlM9,putonice.Place5mlcold60%sucroseina15mlpolypropelenetube.ShakethewormsandcarefullylayerontopofthesucrosewithapasteurPipette.Spininaclinicalcentrifugeat4°for5".Removethetoplayer,whichcontainscleanhealthyworms,toanewtube,addsomemoreM9towash,andpellettheworms.

5.Washandpelletthewormsin4mlTEN.Removethesupernatant,resuspendtoatotalvolumeof.5ml(note:uselessthan~200µlwormshere,ifyouhavemorethensplitintotwotubes.Shouldget200µlwormsfrom1-3largeplates).Movetoa1.5mlEppendorftube.Atthispoint,canthrowthewormsintothe-20°freezertosynchronizeprepsofdifferentstrains.Freezing/thawingherehelpsthefollowingproteinaseKdigestions,andsoisusefulevenifyoudon"twanttostoretheworms.

6.Tothewormsin500µlTENina1.5mleppendorftubeadd:

25µl10%SDS(finalconc.0.5%)

2.5µl20mg/mlProteinaseK(Imakethisfresh)(finalconc.0.1mg/ml)

1.0µlbeta-mercaptoethanol

-Incubateina50to60°waterbath;gentlyresuspendbyinversionat1",3",10"toachievea~uniformsuspensioninsteadofonebigglopfloatinginliquid.

-Afteronehouraddanother2.5µlproteinaseK,andputbackinthewaterbath.

-Afterthesecondhour,addanother2.5µlproteinaseK,putbackinthewaterbath.

-Attheendofthethirdhour,youshouldhaveauniformnotveryviscousmilkyyellowsolution.Ifthereisstillgloppywormgooppresent,canletthedigestiongoovernight.

7.Phenol/chloroformextractbyadding0.5ml50/50phenol/chloroform(isoamylalchohol).TheHorvitzlabusesacommercialpreparationofthisfromAmericanBioanylytical(#AB1605).Atthisstepandallsubsequentones,treattheDNAverygentlytoavoidshearing.Mixtheaqueousandphenolphasesbygentleinversion.Thencentrifugefor2minatroomtemp.Transfertheupperaqueousphasetoanew2mleppendorftubeusingacutoff200µltip(beclean,notgreedy).Toincreasetheyield,reextractthephenol/CIAphasewith200µlTEN,andcombinethetwoaqueoussups.Iftheproteininterfacewasextensive,canrepeatthephenol/CIAextraction.

8.Add1.2mlEtOHatroomtemp,andmixbyinvertinggently,andflickingthetubetoproduceagentleswirlingthatwindstheDNAstrands.ShouldgetaprecipitatedclotofDNAalmostimmediately.Donotspinthetube:insteadpicktheDNAclotoutwitha200µltiponapipetteman(withoutsucking)andplaceinatubeof70%icecoldEtOHtowash.ThisgivescleanerDNAthanpelleting.(Ifnoppt.isvisIBLe,youcanspinthetube,buttheyieldofDNAisprobablypoor).Spinthe70%tubeverybriefly(10"),removethesupernatant,anddrythepelletonlyverybriefly;wantittoremainmoistwith70%EtOHforeasierresuspension.

9.Resuspendthepelletin0.5mlTEN.Thisisadifficultresuspensionandmayrequirethefollowingsequence:

-Letthepelletrehydrateat4°afewhourstoovernight.

-Dissolvethepelletmorebyputtingat50-60°for1-2hours.Atthisstepyoumayacceleratetheresuspensionbybreakingthepelletupusingapipettemananda200µltiptogentlysuckupandspitoutthepellet,breakingitintosmallpieces.ThistreatmentappearsnottosheartheDNAenoughtoshowupintheSoutherns.

-Becareful,becausethepelletoftenbecomescrystalclearafterrehydrating,andcanonlybeseenduetoitsrefraction.Again,beverygentle(novortexing)soasnottosheartheDNA.

10.Add3µl10mg/mlboiledRNaseA(storethisstockfrozen)(finalconc.40µg/ml).Incubate37°for1-2hour.Phenol/CIAextractasbefore.Chloroformextract(Ioftendon"tuseisoamylalchoholhere).Add2volumes(~0.8ml)100%EtOHatroomtemp.Invertandflickasbefore,andagainpickthepelletoutwithapipettetiptoatubeoficecold70%EtOH.ThenucleicacidprecipitatewillbesubstantiallysmallerthistimeduetotheabsenceoftheRNA.Letthe70%tubesitatroomtemp.for1hourtoremovesalts.Spin10",removethesup,drythepellet,andresuspendinTE(~100µlperlargeplateofworms).Again,thisresuspensionisdifficultandmayrequirerehydratingovernightat4°followedbyacouplehoursat50-60°.Ifthepelletstillwon"tresuspend,tryaddingmoreTE.

Solutions:

TEN:20mMTrispH7.5,50mMEDTA,100mMNaCl

II.RestrictiondigestionofwormgenomicDNA:takesovernight

AchievingcompletedigestsofgenomicDNAisproblematicinsomepeople"shands.Toensurecompletedigests,tryusing10unitsofrestrictionenzymeperµgofDNAanddigestingovernight(note:thisisonlyusefulforstablerestrictionenzymes-theBiolabscataloghasatableshowingthestABIlityofvariousenzymes).EcoRIisexcellentatcuttinggenomicDNA,HindIIIisgood,othersvary.Afterdigestion,runanaliquotonananalyticalgeltocheckthedigestion:shouldseeasmearwithabell-shapeddistributionofintensity.Theaveragesizevariesaccordingtothelengthoftherecognitionsequence,andalsotheGCcontentoftherecognitionsite;ATrichsitesoccurmorefrequently.Youdon"twanttoseeDNAhangingupatthemobilitylimitofthegel;youdowanttoseeafewtightbandswithinthesmear(thesearerepetitivesequences).EcoRVandXbaIareother6-cutterswithAT-richrecognitionsitesthatworkokay.

Othertricksforachievingcompletedigests:somepeopleroutinelyadd5mMspermidinetotheirdigests,whichreputedlysoaksupinhibitorsofrestrictionendonucleases.Anotherforsurehelpistodilutethedigestintoalargervolume(thusdilutingtheenzymeinhibitors)andthenprecipitateandresuspendthecutDNAinthedesiredvolume.Youshouldbeabletodigest5µgDNAina20µldigestwithcleanDNA,butdiluting10-foldwillhelpifthisdoesn"twork.ForgenomicDNApreparedasabove,thesetricksaren"tnecessaryforEcoRIorHindIIIdigests.

III.Runningthegel;takesovernight

Typically,wormDNAiscutwithasix-cutterenzymeandawholecosmidorTc1probeisused.Inthesecases,manybandsinthe~1-10kbrangewillbedetected,andthusitisimportanttoachievegoodseparationoffragmentsinthisrange.

Typicalgelconditions:usea35cmlongx15cmwidegelbox.Thegelpouredinsideis23cmlong.Poura0.8%agarosegelin1XTAE,withEtBRinthegelandrunningbuffer.Don"tpourthegelanythickerthannecessary;thiswilljustinterferewiththetransfer.Usea20wellcomb(eachwellis4mmwide).Runthegelovernight(11-13hours)at125volts,recirculatingthebufferslowlywithaperistalticpump(weuse"masterflex"pumpsfromColeParmerInstruments).Runthepumpaboutasslowlyasitgoes;maywanttoruntheDNAinforanhourorsobeforeturningthepumponsoasnottodisturbtheDNAinthewells.(Ifyourunthepumptoofastthereisadangerthatthegelwillbefloatedofftothesideofthegelboxbytheflowofthebuffer!)Load1-5µgDNAperlane.UsingthelargeOwlScientificboxesIpoura300mlgel,andittakes13hourstorunthebromophenolbluetothebottomofthegel.

Toachieveasignificantincreaseinseparationinthe1-10kbrange,runthegelusingafieldinverterboxbetweenthepowersupplyandthegelbox.(Notethatthecheapblue"BlairCraftScientific"powersuppliesdon"treallysupplysteadydirectcurrentandshouldn"tbeusedwiththefieldinverter;usetheBioradorHoefferDCpowersupplies).WeusetheMJResearch"ProgrammablePowerInverter"modelPPI-200withthefollowingparameters:

A=0.1B=0.01C=0.3D=0.03E=11F=0G=0

Plugtheleadsfromthepowerinverterintothepowersupply,andconnectleadsfromthegelboxtothefemalesocketsintheinverterbox.Canruntwogelsinparallel(notseries!)offofoneinverterbox/powerinverter.Aftersettingandcheckingtheparameters,turnthepowersupplyto125volts,andthenremembertopressenterontheinvertertobegintheinversionprogram.

IV.Southerntransfer;takesafewhourstoprocessthegel,overnighttransfer,andacouplehoursthenextdaytobaketheblot.

Afterthegelisrun,takeapictureontheUVlightboxwitharulerlaidnexttothegel.Later,willusethistomeasureoutthesizeofthebandsontheSouthernblot.Icutoffthelowerleftcornerofthegeltohelpremembertheorientation.Whileatthelightbox,leavethegelonthe250nmlightboxwiththelightonfor1min.ThisnickstheDNAandallowsmoreefficienttransferofhighmolecularweightfragmentsoutofthegel.(Note:don"tleaveitontheUVboxtoolong:5minutesnickstheDNAsomuchthatitwillbeintinypiecesthatwilldiffuseinthegelgivingfuzzybandsontheblot.)

Insubsequentsteps,handlethegelverygentlytoavoidbreakingit.Soakthegelinseveralvolumesofdenaturingsolutionfor2X15"withgentleagitation.Rinsebrieflywithneutralizingsolution,andsoak2X30"inseveralvolumesneutralizingsolution.

Cut~4sheetsofWhatmann3MMorequivalentandasheetofNytran(SchleicherandSchuel)tothesizeofthegel.AlwayshandletheNytranwithcleangloves.CutoffthecorneroftheNytranthatwillbeoverthelowerleftcornerofthegeltolaterorienttheblot.Nytranispreferredovernitrocellulosebecauseitdoesn"ttearasreADIly.Wetthe3MMinadishof20XSSPE.Nytranisbestwettedfirstindistilledwater,andthentransferredto20XSSPE.Placecommercialkitchensponge(s)inatray;useseveralspongessidebysideifthegelisbiggerthanonesponge.Fillthetryupto~1cmbelowthetopofthespongeswith20XSSPE.Laytwosheetswet3MMontop;smooththemdowntoremoveairbubbles.Laythegelupsidedown(i.e.wellsdown)onthe3MM,smoothouttheairbubbleswithawetglovedfinger.PlacestripsofParafilmalongeachsideofthegel;thesewillstopbufferfromwickingupexceptthroughthegel.LaytheNytranonthegelwiththenotchedcorneroftheNytranmatchingthenotchedcornerofthegel.Smoothoutairbubbles.DonotadjusttheNytranonceitisplacedonthegel.Laytwosheetsofwet3MMontop;smoothoutthebubbles.Layseveralinchesofdrypapertowelsontop.Placeaglassplateorequivalentontopand~200gmweighttoensureevencontactwithinthestackoftowels.Waitovernight;donotdisturbduringthetransfer.

Thenextmorning,removethepapertowelsandupper3MMsheets,butdonotremovetheNytran!BeforeremovingtheNytran,useadullsoftpenciltomarkthepositionofthewellsontheNytran.Ballpointpenalsoworks,butthemarksmayrunalittle.ThenremovetheNytran;placebetweentwosheetsofdry3MM.Thegelshouldbeadrysmashedflatrubberythingatthispoint.Aftertheblotairdries(~15min),bakeitinavacuumovenat80°for2hours.

SomepeopleUVcrosslinktheDNAtotheNytran.CrosslinkwhiletheblotisstillmoistinaStratalinker;rinsetheblotin2XSSPE,daboffexcessliquidon3MMpaper,placethemoistfilterinthemachineDNAsideup,presstheonbutton,presstheautocrosslinkbutton,pressthestartbutton.ThemachinecountsdownasitmonitorstheUVtogivetheoptimaldose.Thentaketheblotout,turnthepoweroffwiththepowerbutton,andbaketheblot.Ofcourse,bakingisn"tactuallynecessaryaftercrosslinking,butyoucandoitifyouwant.

Solutions:

Denaturingsolution:0.5MNaOH,1.5MNaCl

Neutralizingsolution:1MTrispH8,1.5MNaCl

20XSSPE:175.3gNaCl,27.6gNaH2PO4.H20,7.4gEDTA:dissolvein~800mlH20,adjusttopH7.4w/~6.5ml10NNaOH.Adjustvolumeto1liter.

V.Probingtheblot;afewhourstomaketheprobeandprehyb,overnighttoincubateblotandprobe,afewhourstowashthenextday,1-4daystoexposetheautorad.

1.Labellingtheprobe-obviously,followallthenormalradioactivitysafetyprecautions

-Ifyoustoreitfrozen,takethealpha32P-dATPoutofthefreezertothaw.

-Put50ngDNAtobelabelledinatotalvolumeof18.9µlH20.Thisisusually1-4µlofatypicalcosmidprep.ItisunnecessarytocutcosmidDNApriortolabelling.Forlabellingfragmentsfromarestrictiondigest:Iprefernottodothelabellinginthelowmeltagarose,sincetheagarosesomewhatinhibitsthelabellingreactionandthespecificactivityoftheresultingprobeisonlymarginallyadequateforgenomicSoutherns.Itiseasytopurifythefragmenteitherusingagarase,orbyadsorptiontoglassasinthecommercialQiexkit,andthiswillincreasetheefficiencyoflabellingconsiderably.

-BoiltheDNAtodenatureitforafewminutes.Brieflyspinthetubeandputonicetocool.Add6.6µl5XOLB,5µlhotdATP(50µCi),and2µlKlenow.

-Allowthelabellingreactiontoproceedatroomtemp30mintoovernight.Thereactionappearstogotocompletionby45min.

-Somepeopleusetheentirereactionintheirhybridization.However,anyoneintelligentwillrunaspincolumnatthispointandmeasuretheamountofcountsincorporated,sincethisisthemainfactordeterminingthestrengthofsignalontheautorad.Addabout20µlof50mMEDTAtostopthereactionandrunaspincolumnasusual.Expectabout50%ofthecountstobeincorporatedforanaveragelabelling.

2.Prehybridizationoftheblot-candothiswhiletheprobelabels.

Wettheblotinatrayof6XSSPE.Makesomeprehybridizationbuffer.Puttheblotinahybridizationtube(orasealamealbag),andaddthebuffer(theoretically~0.5mlpersquareinchofblot,inpracticeIuse~30mlsforalargeHybaidtube),andincubateat65°foracouplehoursorlonger.Ihighlyrecommendusingthe"Hybaid"hybridizationovenwiththerollerinitforglasstubes.Itmaintainstemperatureaccurately,andminimizesyourexposuretoradioactivityandchanceofhavingaspill.Togettheblotsintothetubes,wetthemfirst,fillthetube~1/3fullof6XSSPE,anduseaglassrod(aThermometerwilldoifyouareverycareful)tohelpshovetheblotintothetubeandtoplasteritagainstthewallofthetube.Whenusingglasshybridizationtubes,don"ttightenthecapstoomuch:theytightenupintheoven.MakesurethecapofthetubehasaredrubberOringinit;theothertypeofcapcanleak.

3.Hybridization:pourtheprehybsol"nout.Addhybridizationsolution(Iuse20mlsforalargeHybaidtube)totheblot.DenaturetheprobeDNAbyA)heatingto100°Cfor5-10min,orB)addNaOHto0.2M,incubate5"roomtemp(theSSPEwillbufferthisoutwhenyouaddittothehybsolution).Addtheprobetotheblot/hyb.solution,closethetube/bag,andincubateovernightat65°.

4.Posthybridization:Make1literofposthyb.washsolution,andheatashakingwaterbathto65°.Pouroutthehybsolutionintoa50mlplasticdisposablecentrifugetubeifitistobereused(probeisgoodforacoupleofweeks,andmustbeboiled5minandchilledonicebeforereuse).Rinsetheblot(inthetube)brieflywithsomewashsolution.Removetheblottoatupperwaredish.Addagenerousamount(afewhundredmls)ofwashsolution.Washtheblot3X30"inthe65°waterbath.Atthispointthegeigercountershouldnotdetectcountsontheblotifyouarejustprobingforsinglecopygenomicsequences.WhenprobingwithTC1orotherrepetitivesequenceprobes,thesignalmaybehotenoughtodetectwiththegeigercounter.

Dabthebloton3MMpapertogetridofexcessliquid,wrapthemoistblotinSaranwrap,tapetoanoldautorad,applyastripofphosphorescentMarkernexttoit(Stratagene),andtakeanexposurewithanintensifyingscreenat-80°.SinglecopygenomicSouthernsrequireovernightto4dayexposureswithwholecosmidprobes.I"vegottenblotsthatrequireonly1hourexposuresusinglabelledrestrictionfragmentprobes.Alwayskeeptheblotwetandfrozen,sothatitcanbestrippedandreused.

Solutions:

5XOLB:(oligonucleotidelabellingbuffer)

250mMTrispH8.0250µl1M

25mMMgCl225µl1M

5mMß-mercaptoethanol0.35µl14.4Mstock

2mMdCTP20µl100mMstock

2mMdGTP20µl100mMstock

2mMdTTP20µl100mMstock

1MHEPESpH6.6500µl2M(2.38g+4mlH20,adjustto6.6w/NaOH,adjustvolto5ml)

1mg/mloligonucleotides(randomprimers:Pharmacia#27-2166-01)

H20to1ml

Prehybridizationsolution:notethatsomeleaveouttheDenhardt"s

30ml20XSSPE

1mlboiled10mg/mlsalmonspermDNA(storefrozenin1mlaliquots)

10ml50XDenhardt"ssolutions(seebelow)

5ml10%SDS(addsomeH20firsttopreventthisfromppting)

H20to100mls

Hybridizationsolution:Sameasprehybsol"naboveexceptwithouttheDenhardt"s

50XDenhardt"ssolution:

5gFicoll

5gpolyvinylpyrrolidone

5gBSA(pentaxfractionV)

500mlH20

Mixovernight.Thenfilterthruadisposablefilterunitandfreezein10mlaliquots.Alternatively,addEDTAto5mMandstoreat4°.

Posthybridizationwashsolution:0.2XSSPE,0.5%SDS

Recipe:10ml20XSSPE,940mlH2O,50ml10%SDS

VI.Strippingtheblotforreprobing.

Hybridizedradioactiveprobescanbestrippedofftheblotandtheblotreprobedseveraltimes.Alternatively,iftheblothassatinthefreezerforseveralmonthstothepointwheretheradioactivesignalhasdecayedtoaninsignificantlevel,itcanbereprobeddirectlywithoutstripping.

1.Prepare3erlenmeyerflaskseachwith500mlof0.05xSSPE,0.01MEDTA

Recipe:Add1.25ml20xSSPEand10mlof0.5MEDTA(pH8.0)to500mldH20.

2.Heatfirstflaskinmicrowaveoven6mintoboiling.ImmediatelyaddSDStofinalconcentrationof0.1%(5ml10%SDS),andgentlyplacetheblotintothehotsolution(usingaglassrodtopushitinhelps).

3.Puttheflaskina65°waterbathfor15min.

4.Repeatsteps2and3withthenexttwoflasksofstrippingsolution.

5.Rinsebrieflyin0.01xSSPEatroomtemp(0.25ml20xSSPEin500mldH20).

6.Removeexcessliquidbydabbingthebloton3MMpaper.WrapinSaranwrap,andexposeovernighttocheckfortheabsenceofradioactivity.

7.Toreusetheblot,prehybandhyb.asusual.

Warning:Probesirreversiblybindtothefilterifitiseverallowedtodry.Therefore,makesuretheblotisalwaysmoistatallstagesduringhybridization,washing,exposuretofilm,andstorage,ifyouwanttoreuseit.

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