Grow10mlYPDcultureso/n.Figureoutcelldensity;inoculate30mlYPDandgrowo/nsothatcelldensityisapproximately2x10⁸cells/mlthenextmorning.Spindowncellsin50mlsterileconicaltubesfor10min.Centrifugationstepsareperformedat4degrees;allotherstepsarecarriedoutatrm.temp.unlessotherwisespecified.ResUSPendcellsin10mlwaterandspindowncells.Resuspendcellsin3mlof0.9Msorbitol,0.1MEDTA,50mMDTT,pH7.5.Add0.25mgZymolyasedissolvedin200ul0.9Msorbitolandincubatewithoccasionalshakingat37degrees.Conversionofspheroplaststakes15-120min.dependingonthestrainused,onthegrowthmedium,andonthegrowthphase.Checkforspheroplastformationbymixing4ulcellsand4ul0.1%SDSonamicroscopeslide.Formationiscompletewhen80-90%ofthecellsare"ghost"cells.Comparetoaslidewith4ulcellsand4ulsorbitolsolution.Spinspheroplastsfor5min.andcarefullydiscardthesupernatant.Resuspendspheroplastsin3mlof50mMTris-HCl,50mMEDTA,pH8.0,byslowlyandrepeatedlydrawingthespheroplastsintoaPipette.Thenmixwith0.3ml10%SDSandincubateat65degreesfor30min.Add1ml5MKOAc,mix,andletsitonicefor60min.orlonger.Thewhiteprecipitatethatformsconsistsmainlyofinsolublepotassiumdodecylsulfateanddenaturedproteins.Transfertoa50mlcentrifugetubeandspinat15,000rpmfor30min.inaSorvallSS34rotor.Transfersupernatant(about4ml)toa12mldisposablecentrifugetube.Add4mlice-coldabsoluteethanol.Onmixing,thenucleicacids(2%DNAand98%RNA)andsomeresidualproteinswithimmediatelyprecipitate.Spinat10,000rpmfor10min.anddiscardthesupernatant.Washwith4ml70%ethanol.Spinat10,000rpmfor10min.

Resuspendin300ulTE,pH7.5.Pelletwilltakeawhiletodissolve.A10min.incubationat42degreescanhelp.MayneedtoletsitO/Ninfridge.Atthispointkeepa3ulaliquottocomparetoprepafterRnasetreatment.Add15ul10mg/mlDnase-freeRnaseandincubateat37degreesfor30min.ThestockofRnaseisdissolvedin10mMsodiumacetate,pH7.0,andkeptat–20degrees.Add300ulphenol/chloroform(1:1)andmixbyinverting.Spinfor10min.Transfersupernatanttonewtubes.Add15ul3MNaOAcand900ulisopropanol.Ppt.shouldbeimmediatelyvisIBLe,ifnot,putondryicefor5min.Spinfor5-10min.Washwith80%EtOH.Airdrypellet.Resuspendin100-300ulTE,pH7.5.RunanaliquotofpurifiedDNAandthealiquotsofDNAbeforeRNasetreatmentona0.7%agarosegel.TheDNAshouldbestoredat+4degreesor–70degrees,notat–20degrees.Frequentfreezingandthawingshouldbeavoided.

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