1.ShakeE.coliharboringplasmidat37Covernightin50mlofTBcontainingappropriateantibiotics.(whenusingampicillin,additionoftheantibioticsto100-200ug/mlratherthanusual50ug/mlmayimprovetheyieldofplasmids.) 2.Spinthebacterialcultureat6,000rpmfor10minat4C.Discardthesupernatant. 3.(0ptional)SUSPendthecellinabout20mlofdeionizedwater.Spinagain.Discardthesupernatant.Removeallofthesupernatantfluidusingpipetman. 4.Resuspendthepelletin5mlofSolutionI. 5.Add10mlofSolutionII.Mixwellbyinvertingthebottlemorethan10times.ThesolutionshouldbeclearaftermixingSolutionII. 6.Add7.5mlofSolutionIII.Mixwellasabove. 7.Spinthelysateat10,000rpmfor10minat4C. 8.Transferthesupernatantintoanewbottle.Addabout15mlofisopropanol,mixwell,andstorethebottlefor10minatroomtemperature. 9.Spinat10,000rpmfor10minat4C.Discardthesupernatant.Removeallofthefluidusingpipetman. 10.Dissolvethepelletin600ulofTE.Transferthesolutionintoamicrofugetube. 11.Add200ulof8MLiCl.Mixwell,andthenspinthesolutionat14,000rpmfor5minat4C. 12.TransferthesupernatantcontainingplasmidDNAtoanewmicrofugetube.Add600ulofisopropanol.Mixwell,andthenspinthesolutionat14,000rpmfor5minat4C. 13.Discardthesupernatant.Rincethepelletandthewallofthetubewith500ulofcold70%ethanol.Discardthefluid. 14.Addtothepellet400ulofTEcontainingDNase-freeRNaseA(20ug/ml).Incubatethetubefor30minat37C. 15.After30min,carefullycheckthecontentofthetube.IfnucleicacidpelletisvisIBLeatthebottomofthetube,vortexwelltodissolvethepellet.Incubatethetubeat37Cforfurther30min. 16.Add240ulof2MNaCl,20%PEG8000.(PEG6000suppliedfromJapanesesuppliersisessentiallyequivalenttoPEG8000,andworkswell.) 17.Spinat14,000rpmfor5min.Discardthesupernatant.Rinsethepelletwith300ulofcold70%ethanol.Discarthefluid.Dissolvethepelletin400ulofTE.(Optional:RepeatPEGprecipitationoncemore.ThisisrecommendedforpreparingdephosphorylatedlinearvectorsincetraceamountofshortRNAinthevectorpreparationmayinterferewiththedephosphorylationreaction.) 18.ExtracttheplasmidsolutionwithchloroformtoremovePEG.Takeaquaousphase.Extracttheaquaousphasewithphenol.Takeaquaousphase.(Optional:Repeatphenolextractionuntilnointerphaseisvisible.Thisisrecommendedforpreparingthetemplateforinvitrotranscription.)Extracttheaquaousphasewithchloroformorethylethertoremovetraceamountofphenoldissolvedinthesolution.Takeaquaousphase(orremoveorganicphase). 19.Add0.1volumeof3Msodiumacetateand3volumeofethanolintotheplasmidsolution.Spinat14,000rpmfor5minat4C.Discardthesupernatant.Rinsethepelletwith200-500ulof70%ethanol. 20.Storetheopentubeonthebenchuntilthevisibletracesofethanolhaveevaporated. 21.DissolvetheDNApelletin200-500ulofTE.Typically300-800ugofplasmidshouldbeobtainediftheplasmidhavepUC-basedreplicationorigin. SOLUTIONS SolutionI 50mMglucose25mMTris-Cl(pH8.0)10mMEDTA(pH8.0)Autoclave,andstoreatroomtemperature. SolutionII 0.2NNaOH1%SDSStoreatroomtemperatureinaplasticbottle.Don"tautoclave. SolutionIII 5Mpotassiumacetate60mlglacialaceticacid11.5mlDistilledwater28.5mlStoreatroomtemperature.Thissolutioncanbeautoclaved.However,weusuallyusethissolutionwithoutautclaving.PurificationofPlasmidfrom50ml-culture