Phenol/FreezeRNAPrep

Schmittetal.(1990)NAR18,3091-3092.

DEPCisusedtoridsolutionsofRNases.DEPCisverytoxicandshouldbeusedonlyinthehood.Stuffyouneed:(DEPCtreatmentmeans:addDEPCto0.1%,mixO/N,autoclavetodestroyDEPC)DEPCtreatedwaterAEbuffer(DEPCtreat)50mMNaacetate,pH5.310mMEDTA10%SDSPhenol,equilibratedwithAEbufferPhenol:chloroform:isoamylalcohol,25:24:13MNaacetatepH5.3(DEPCtreat)RNase-free95%EtOH(-20degC)RNase-free80%EtOH1.Grow10mlofyeastto5E6cells/ml.2.HarvestcellsbycentrifugationandresUSPendcellsin400µlofAEbuffer.3.Transfercellstoa1.5mlmicrocentrifugetubeandadd40µlof10%SDS.Vortex.4.Addequalvolumeofphenol,vortex,andincubateat65degCfor4minutes.5.Rapidlychilltubeindryice/ethanolbathuntilphenolcrystalsappear,andcentrifugeatroomtemperaturefor2minutes.6.Transferaqueousphasetoanewtube,addanequalvolumeofphenol/chloroform/IAA,vortex,andspinfor5minutesatroomtemperature.7.Transferaqueousphasetoanewtube,add1/10volumeof3MNaAcetate,pH5.3,2.5volumesofethanol,andthenprecipitatefor20minutesat-20degC.8.PelletRNA,washwith80%ethanol,airdrypellet,andresuspendin20µlwater.

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GlassBeadRNAPrep

DEPCisusedtoridsolutionsofRNases.DEPCisverytoxicandshouldbeusedonlyinthehood.Stuffyouneed:(DEPCtreatmentmeans:addDEPCto0.1%,mixO/N,autoclavetodestroyDEPC)DEPCtreatedwaterRNAextractionbuffer0.1MNaCl(DEPCtreat)10mMEDTA(DEPCtreat)5%SDS(DEPCtreat)50mMTris-HCl,pH7.5(CANNOTDEPCtreat)PCI50:50:1ofphenol:chloroform:isoamylalcoholSEVAG24:1chloroform:isoamylalcohol3MNaOAcpH5.2(DEPCtreat)RNase-free95%EtOH(-20degC)RNase-free70%EtOH1.Growcellstonolaterthanlatelogphase(1E7cells)in20mlofmedia(protocolcanbescaledupordown).2.Transfercellsto50mlFalcontube.Spin3000rpminBeckmantabletopfor5min.3.WashpelletwithDEPCtreatedwater.4.Spin3000rpm5min.Canstorepelletat-70degCindefinitely.5.Resuspendpelletin0.6mlRNAextractionbuffer.6.IMMEDIATELYaddanequalvolumeofPCI.Mix.7.LetsitatRT5-6min.8.Duringthistime,transferto13x100mmglasstubes.9.Addglassbeadsto~3/4uptoorganicphasemeniscus.Vortex2minatmaxspeed.10.Transfersolutionto1.5mlmicrofugetube.Separatephasesbycentrifugation.11.Extractaqueousphase2XwithPCI,then1XwithSEVAG.(Addequalvolumeseachtime).12.Add0.1volume(approx.40µl)3MNaOAcpH5.2and2-3volscold95%EtOH.Ppt.at-20degCfor1hr.13.Pellet,andwashpelletwith70%EtOH.14.ResuspendwithDEPCtreatedwater(~30µl).Storeat-70degC.

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