Procedure1.Grind2to5goffrozenleavestoaveryfinepowderwithaliquidnitrogen-cooledmortarandpestle.2.Add25mlofCTABBufferandtransfertoa50mltube.3.Incubateat65°Cfor20minwithoccasionalvigorousshaking.4.Add10mlofChloroform,shakewell,andplaceonatubeinverteratroomtemperaturefor20min.5.Centrifugeat1,000Xgfor5mintoresolvephases.6.Transfertheaqueousphasetoafreshtube,add17mlofIsopropanol,mix,andplaceonicefor10min.7.Centrifugeat1,000Xgfor5mintocollecttheprecipitate.8.Discardthesupernatantanddrytheinsideofthetubewithapapertowel(donotdrythepellet).9.Add4mlofTEBufferanddissolvetheprecipitatebygentleinversion.10.Add4mlof4MLithiumAcetateandincubateonicefor20min.11.Centrifugeat1,000Xgfor10min.12.Transferthesupernatanttoafreshtube,add16mlof100%Ethanol,andincubateonicefor20min.13.Centrifugeat1,000Xgfor5mintocollecttheprecipitate.14.Discardthesupernatantanddrytheinsideofthetubewithapapertowel(donotdrythepellet).15.DissolveDNAin900μlofTEBufferbygentlepipetting.16.Add100μlof3MSodiumAcetate.Dividethesampleevenlyintotwomicrocentrifugetubes.17.AddanequalvolumeofPhenoltoeachtube,mixwell,centrifugetoseparatethephases(1,000Xg),andsavetheaqueousphase(upperphase).18.AddanequalvolumeofPhenol:Chloroformtoeachtube,mixwell,centrifugetoseparatethephases(1,000Xg),andsavetheaqueousphase(upperphase).19.AddanequalvolumeofChloroformtoeachtube,mixwell,centrifugetoseparatethephases(1,000Xg),andsavetheaqueousphase(upperphase).20.Add2volumesof100%Ethanoltoeachtubeandincubateonicefor5min.21.Centrifuge5mintocollecttheprecipitate(approximately5,000Xg).22.Discardthesupernatant.23.Centrifugefor5additionalminandremoveasmuchoftheremainingliquidaspossIBLewithaPipette.Donotdrythepellet.23.Add100to250μlTEBuffer.Dissolvethepelletbygentlepipetting.Solutions

Phenol:Chloroform		1:1Phenol:Chloroform
4MLithiumAcetate
3MSodiumAcetate
TEBuffer		1mMEDTA,pH8.010mMTris
CTABBuffer		800mMNaCl1%(v/v)Sarkosyl140mMSorbitolAutoclave22mMEDTA220mMTris,pH8.00.8%(v/v)CTAB