ISOLATIONOFDNAFROMAGAROSEGELSWITHDEAEPAPER MATERIALS: TheDEAEpapercanbeusedassuppliedbySchleicherandSchuell,howeverthebindingcapacitycanbeincreasedbywashingthepaperin10mMEDTA,pH7.5for10minutes,0.5MNaOHfor5minutes,followedbyseveralrinsesindistilledwater.Thepapercanbestoredat4oCforseveralmonths. NETbuffer: 0.15MNaCl 0.1mMEDTA 20mMTris-HCl,pH8.0 HighsaltNETbuffer: 1.0MNaCl 0.1mMEDTA 20mMTris-HCl,pH8.0 METHOD: AftertheDNAbandshavebeenseparatedelectrophoretically(ataround80v),thegelisviewedunderlongwaveUVlightandaslitcutinthegel,justaheadofthedesiredband.ApieceofDEAEpaperisinsertedintheslit.AnotherpieceofDEAEpapercanbeinsertedbehindthedesiredband,topreventunwantedbandsbeingtrappedbythepaper. Electrophoresisiscontinuedforapproximately10to15minuteswiththevoltageincreasedto100-120V. TheDEAEpaperisremovedandbindingoftheDNAcheckedbyviewingthepaperwithlongwaveUVlight.ThepaperisthenplacedinanEppendorftubecontaining500ulNETbuffertowashawayremainingagarose.TheboundDNAcannowbestoredat4oCforseveraldays.TheDNAshouldalwaysremaincoveredwithbuffertopreventirreversIBLebindingwhichmayoccurifthepaperisallowedtodryout. ToelutetheboundDNA,placetheDEAEpaperinaneppendorftubecontaining250ulofhighsaltNETbuffersothatthepaperiscompletelycovered.MakesurethatthesideofthepapercontainingtheDNAfacesthebufferandnotthesideofthetube.Ifnecessary,centrifugebrieflytosubmergethepaper. Incubatethetubeat55-68oCfor45minuteswithoccassionalswirling(oruntilalltheDNAhasbeenelutedascheckedunderlongwaveuvlight). Removethebuffertoafreshtubeandadd50ulhighsaltNETbuffertowashthemembrane.Removetheadditionalbufferandaddtothefreshtube. Centrifugethebufferfor30secondstopelletanyremainingDEAEpaperfibresandremovethebuffertoafreshtube(leavingapprox.20ulinthebottomofthetube). Extracttheresidualethidiumbromidewith3volumesofwatersaturatedbutanol. Removeandretainthebottomlayerandadd1/20volumeof3.0Msodiumacetate,pH5.25and2.5volumesofcoldabsoluteethanol.Incubateat-70oCforatleastonehour.Ethanolprecipitateandvacuumdry.
