Solutions

Protocol:Digest5-10μggenomicDNAovernightwithrestrictionenzymeofchoice.RundigestedgDNAon0.8%TAEgelwithMarker(withnoethidiumbromide)

TransferSetup:1.RemovegelandplaceintonewlymadeEtBrsolutionandrockforapproximately10min.2.RinseinH₂Oandtakephotoofgel3.UVirrADIategelfor1.5minontransIlluminator4.Slowlyrockgelfor30mininDenaturationSolution5.RinseinH₂Oandslowlyrockgelfor30mininNeutralizingSolution6.Preparewick:cuttwopiecesofWhatmanpaperwideenoughtoaccommodategelandlongenoughtoextendintoblottransferdishcontaining20XSSC7.Rinsegel,flipandplaceonWhatmanpaper(thisshouldbetheonlytimethegelisflipped)8.Cutnylon(Schleicher&SchuellNytran),prewetinH2Oandplaceontogelmakingsurethatnoairbubblesaretrappedbetweennylonandgel9.Stackgelblottingpads(prewetfirstonein20XSSC).10.Applyweighttostackandallowtotransferovernight(setupshouldhaveapyramidshape)

Random-PrimedLabelingofDNAFragment:Forprobesynthesis,youcanuseacommerciallyavailablerandom-primedlabelingkitusingα-32P-dCTP(i.e.,Roche)orDIG-labeledprobe(Roche).Or,doyourrandomprimedlabelingtheold-fashionedway:(1)CombineinEppendorftube:Incubate95°Cforapproximately5minutes.Spindownbriefly.Putoniceimmediatelytocool.(2)ToEppendorfwithDNA&H2Oadd:1μleachdATP,dGTP,dTTP(dilute10mMstocksofindividualnucleotides1:200in10mMTrispH7.5)4μlrandomhexamer(1μg/μlstock=5X)5μl³²P-dCTP2μl10XKlenowbuffer1μlKlenow20μl(3)Incubate30minat37°C(4)AddTEtoincreasevolumeto50μl(5)Runsamplethroughspincolumntoremoveunincorporatednucleotides(i.e.,G-50spincolumns)(6)Count1μlinscintillationcounter(ingeneralyouwanttouseabout50x105cpm,soif1μlofprobeisabout2x105cpm/μl,thereforewanttouse25μl)

Hybridization:1.Takenylonoffgelandrinsebrieflyin2XSSC2.PlaceonapieceofWhatmanpapertoairdry(≈5min)3.UVcrosslinkfor60sec(DNAsidedown)(AutoCrosslinkeravailableinC3-123,TapscottLab)4.Dryin68°Cincubator(0.5–8hrs)5.Placein10mlPre-PreHybsolutioninRobbinstube(orsealedplasticbag)andincubateat68°Cforatleast30min(thisstepcanbeomittedifusingagoodprobethatproduceslowbackground)6.Replacewith10mlPreHybsolution;incubate30mintoovernightat42°C.7.Replacewith10mlHybridizationsolutioncontainingprobe.Hybridizeovernightatappropriatetemperatureusingsufficientprobei.e.,42°C(seeabove).

WashBlot:1.Takeblotoutoftube/bag.Disposeofradioactiveprobe/hybsolutionasradioactivewaste.2.Washbrieflyin3XSSC/0.5%SDS;pouroffliquid.3.Replacewashsolutionandincubateat68°Cfor30min4.Repeatstep35.WrapblotinSaranWrapandplaceonfilm.(ExposeO/Nat-80°C;develop).

StrippingBlot:1.Heat0.1%SDSto100°CinPyrexbakingdish2.AddblottohotSDSsolution3.Turnoffheatandallowtocooltoroomtemp4.WrapblotinSaranWrapandmonitorwithGeigercountertoensurethatblotwaseffectivelystripped