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ISOLATIONOFDNAFROMAGAROSEGELSWITHDEAEPAPER

MATERIALS:

TheDEAEpapercanbeusedassuppliedbySchleicherandSchuell,howeverthebindingcapacitycanbeincreasedbywashingthepaperin10mMEDTA,pH7.5for10minutes,0.5MNaOHfor5minutes,followedbyseveralrinsesindistilledwater.Thepapercanbestoredat4^oCforseveralmonths.

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.TheboundDNAcannowbestoredat4^oCforseveraldays.TheDNAshouldalwaysremaincoveredwithbuffertopreventirreversIBLebindingwhichmayoccurifthepaperisallowedtodryout.

ToelutetheboundDNA,placetheDEAEpaperinaneppendorftubecontaining250ulofhighsaltNETbuffersothatthepaperiscompletelycovered.MakesurethatthesideofthepapercontainingtheDNAfacesthebufferandnotthesideofthetube.Ifnecessary,centrifugebrieflytosubmergethepaper.

Incubatethetubeat55-68^oCfor45minuteswithoccassionalswirling(oruntilalltheDNAhasbeenelutedascheckedunderlongwaveuvlight).

Removethebuffertoafreshtubeandadd50ulhighsaltNETbuffertowashthemembrane.Removetheadditionalbufferandaddtothefreshtube.

Centrifugethebufferfor30secondstopelletanyremainingDEAEpaperfibresandremovethebuffertoafreshtube(leavingapprox.20ulinthebottomofthetube).

Extracttheresidualethidiumbromidewith3volumesofwatersaturatedbutanol.

Removeandretainthebottomlayerandadd1/20volumeof3.0Msodiumacetate,pH5.25and2.5volumesofcoldabsoluteethanol.Incubateat-70^oCforatleastonehour.Ethanolprecipitateandvacuumdry.

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【求助/交流】干扰RNA转染动物 分子生物 综合其他论坛...

【求助/交流】干扰RNA转染动物分子生物综合其他论坛...