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.

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