Northerns

Stuffyouneed:10Xrunningbuffer:50mMNaOAc0.2MMOPSpH7.010mMEDTAfilter,storeindarkatRT,pHto7.0Formaldehydegel:0.9%agarosein1Xrunningbuffer,and1mlformaldehydeper20mlgel.(For200mlgel,dissolve1.8gagarosein170mlwater,letcoolin65degCwaterbath,add10mlformaldehyde(37%stock),and20ml10XMOPSrunningbuffer).1.5XLoADIngbuffer:750祃formamide75祃formaldehyde(37%)150祃10Xrunningbuffer15祃EtBr(5mg/ml)10祃ddH20BestresultsifmadefresheachtimeLoadingDye:Bromophenolbluein35%glyceroland1Xrunningbuffer20XSSC:3MNaCl0.3MNaCitratepH7.01.Pourgelinhood.2.MixRNA(10礸)with1.5Xloadingbuffer(e.g.10祃RNA+20祃LB).3.Incubate5minat65degC.Coolonice.4.Add2祃loadingdye.5.Pre-rungelat5V/cmfor5minutes.Loadsamplesandrun.Mixbuffersafter3hours.6.Washgel2Xinwaterfor15min,then1Xin10XSSCfor15minutes.PhotographgelunderUV.7.Cutnylonmembrane(MSI0.45礽cron#N04HY00010)andseveralpiecesofblotting(e.g.SchleicherandSchuellGB002)papertothesamesizeasthegel.WetthenylonwithdH20,thensoakin5xSSC.8.Assemblesandwich:a.Largesheetofplasticwrapb.Twopiecesblotpaper(precuttosamesizeasgel)soakedin20xSSCc.Gel(wells-sidedown)d.Presoakednylone.Onepieceblotpapersoakedin5xSSCf.10-15piecesofdryblotpaperh.Wrapwholesandwichintheplasticwrapi.Placeglassplateandweightontopandlettransfer>3hr.9.Crosslinkafterblotting.10.Canprobeeitherwithnon-radioactiveprobeorwithradioactiveprobe.