NorthernBlot

I.Electrophoresis

• cleangelboxwithNaOHand/orSDS,2hourstoovernight,rinsewithwater
• prepareAgarosegelsolution[1%Agarose,1xMOPS,H2Oto95%ofendvolume]
• microwaveuntilcompletelydissolved
• cooldownto60-70°C,addFormaldehyde(37%)to0.6Mendconcentration,pourimmediately
• allowgeltohardenatleast30min
• preparerunningbuffer[1xMOPS,0.2MFormaldehyde]

II.Samplepreparation

• use5-10µgtotalRNAperlane(upto30µg)
• bringRNAwithH2ODEPCtoequalvolume(5-10µl),addsamevol.loADIngbuffer
• add0.5µlEtBr(0.5µg/µl)
• heatfor5min@90°C,coolonice

III.Gelrun

• rungel(8x10cm)infumehoodwith70-100V(->50-70mA)
• rununtilBPBisnearthegelend(2.5-3.5h)

IV.NortherntransferofRNA

• soakgel3times5minindistilledwater(toremoveFormaldehyde)
• photograghgelwithrulerbesideit
• cutGeneScreenmembrane(Nylon,DuPont)toexactgelsize
• soakmembraneinwaterforafewseconds
• setupcapillaryblotwith10xSSCtransferbuffer:2wetWhatman-gel-membrane-2wetWhatman-2dryWhatman-papertowel-glasplate-weight
• transfer16-24hwithchangesofthepapertowel
• marklanes,removemembrane,washbrieflyin2xSSC
• placemembraneonwetWhatmanpaperandUV-crosslinkdamp(autocrosslinksetting,254nm,Stratagene,Stratalinker)
• bakemembrane@80°Cfor1-2h

V.Hybridization

• prehybridizemembranefor1-4h@42°Cwith5-10mlprehybridizationbuffer
• heatradioactivelabeledprobefor3min@95°C,coolonice
• discardprehybridizationbuffer,addhybridizationbufferandprobe,incubateON@42°C
• washmembrane1x15minwith2xSSC@RT
• washwith2xSSC,0.1%SDS@65°Cuntilbackgroundislow
• washwith0.1xSSC,0.1xSDS@65°C(optional)
• exposewetmembraneundersaranwrap(-80°C)
• important:neverletthemembranedry(untiltheblotisstripped)

VI.Strippingandre-hybridization

• washmembranefor30minto3hinstripsolution@75-85°Cuntilnoradioactivitycanbedetectedonthemembrane
• membranecannowbeairdriedandstored@RT
• forre-hybridization(upto10times)followthehybridizationprotocol

Buffers:10xMOPS:0.4MMorpholinopropanesulfonicacid(freeacid);0.1MNa-acetate-3xH2O;10mMEDTA;adjusttopH7.2withNaOH;storedarkinfridge:[500ml:41.9gMOPS,6.8gNaAc,10ml0.5MEDTA]LoadingBuffer:1xMOPS;18.5%Formaldehyde;50%Formamide;4%Ficoll400;Bromophenolblue;storeat-20°C:[1ml:100µl10xMOPS,500µlFormamide,185µlFormaldehyde,40mgFicoll400,Bromophenolblue,215µlH2O]Prehybridization-buffer:5xSSC;50%Formamide;5xDenhardt"s-solution;1%SDS;100µg/mlheat-denaturedshearednon-homologousDNA(SalmonspermDNAoryeasttRNA)[100ml:25ml20xSSC,50mlFormamide,5ml100xDenhardt"s,1gSDS,1ml10mg/mlDNA]Hybridization-buffer:Prehybridizationbufferwith5%Dextransulfate(Na-salt,MW500,000,50%stock-solution)andwithoutnon-homologousDNA

100xDenhardt"ssolution:[for500ml:10gFicoll400;10gpolyvinylpyrrolidoneMW360000;10gBSAfractionV;H2O]storeat-20°C.20xSSC:3MNaCl;0.3MNa-citrate[1l:175.3gNaCl,88.2gNaCitrate]Strip-solution:5mMTrispH8;0.2mMEDTA;0.05%Na-pyrophosphate;0.1xDenhardt"ssolution[500ml:2.5ml1MTris,200µl0.5MEDTA,5ml5%NaPP,1ml50xDenhardt"s]