1.Rungel(0.8-1.0%agaroseisbest).ForyeastchromosomalSoutherns,digest20μgDNA.Use50mlminigelformostpurposes.Photographgel,butminimizeexposuretoUVlight.2.Depurination:Placegelin250mls0.25MHCl.(250mls=11mls5.8MHCl250mlsddH₂O)Agitategentlyonrotatorfor10minutesafterdyefrontturnsyellow.3.DecantHCl.WashbrieflywithddH₂O.4.Denaturation:Placegelin250mls1.5MNaCl0.5MNaOH.(500mlsNaCl/NaOH=44gNaCl10gNaOH)Rotategently20minutes.Decant,add250mlsfreshNaCl/NaOH,rotate20minutes.5.DecantNaCl/NaOH.WashbrieflywithddH₂O.6.Neutralization:Placegelin250mlsof3MNaCl0.5MTris,pH7.0.500mls=88gNaCl3gTrisbase36gTrisHClRotategently20minutes.Decant,add250mlsfreshNaCl/Tris,rotate20minutes.7.Measuregelexactly.Cutoffbottomrightcorner.Putgelbackinneutralizationsolution.Cutnitrocellulosepaperabout1mmshorterthangelandwetinddH₂O.Soaknitrocellulosepaper5-10minutesin20XSSC.Cut2sheetsofTHINfilterpaper1mmshorterthannitrocellulosepaper.Cut2sheetsofTHICKpaper1mmshorterthannitrocellulose.Cut2-3inchstackofpapertowels.8.SetupTransfer.Use2piecesofthinfilterpapertomakewick.Placewickoverglassplateonsupportplatform.Removeairbubblesatallstepsinthesetupprocedure.Putgelfacedownonwick.Putwettednitrocellulosepaperdowncorrectlyfirsttime.Putonthinpaperpieceswettedin20XSSC.Assembletransferasdepictedbelow.Use50-100gweightontop.Glassplatesworkwell.Placeplasticwraparoundgelandwicktopreventevaporation.9.Aftertransfer,washfilter5minutesin4XSSC.Removeexcessliquid.Airdryfor30minutes.Bakeat70°Cinvacuumovenfor2hours.Blotcanbestoredundervacuumwrappedinfoil.

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