1.Put1cmtailin1.5mlmicrocentrifugetube. 2.Add500µlTailBufferand30µlProteinaseK.(seerecipesbelow). 3.IncubateO/Nat55°ree;C(or4hoursminimum). 4.Mixwellbyshakingbyrepeatedinversion.DoNOTvortex. 5.Letcooltoroomtemperature. 6.Pre-Spin(togetridoffur)8min(usually4°ree;,TRmok). 7.Poursupernatantintoacleantube(numbered).(Tossoutfurrytube). 8.Add250µlsaturatedNaCl(~6M)andinverttomix(don"tvortex)toprecipitatetheproteins. 9.Letsitfor5minutesatroomtemperature. 10.Centrifugemaxspeedinmicrofuge(~13000rpm),10minat4°ree;C,tospindowntheprotein.(TRmisok,but4°ree;ismuchbetter). 11.Pipetsupernatantintoafreshtube.Avoidtransferringthewhiteprecipitate. 12.Add500µlisopropanol. 13.MixbyinversionuntilawhitethreadymaterialisvisIBLe(DNA!).Shakeuntilthreaddoesn"tgetanybigger,~aminute. 14.Centrifugemaxspeedinmicrofuge(~13000rpm),10min(TRmisok). 15.Pipetoffthesup,beingcarefulnottodisturbthepellet(DNA). 16.Washpelletwith70%EtOH(100-800µl).Vortexbriefly. 17.Centrifuge6minutes. 18.Pipetoffthesup,beingcarefulnottodisturbthepellet(DNA). 19.Tap/Draintubeontoakimwipe. 20.AirDryfor45-60min.(DoNOTuseSpeed-Vac). 21.Dissovethepelletin100µlTris(10mMTris,oruseTE)containingRNase. 22.LetdissolveO/Nat4°ree;C.IMPORTANT-DON"TSKIPTHISSTEP!Mixwell.MeasureOD260orOD260/280.Use20µlforSoutherns,orcheck[DNA]anduse10mg.TailPrepSolutions(non-phenolprep.)RNase:10mg/ml.Use20µRNaseper1mlTris.ProteinaseK(Sigma#63173):10mg/mlinH2O,(activate1hrat37°ree;Cbeforeuse).SaturatedNaCl:AddNaCltoapproximately6M(35g/100ml). ShirleyReynoldsTailPreps:DNAIsolationFromMouseTailsWithoutPhenol
TailBuffer
Stock Reagent Vol.For100mls Final 1M Tris 5mls 50mM 0.5M EDTA 20mls 100mM 10% SDS 5mls 0.5% mQH2O 70mls
