Stuffyouneed:TENSBuffer10mMTris-HClpH8.01mMEDTApH8.00.1NNaOH0.5%SDSTEpH8.010mMTris-HClpH8.01mMEDTApH8.095%EtOHprecooledto-20degC70%EtOHatroomtemperature3MNaAcetatepH5.2Spin1.5mlofovernightculturefor10sinamicrocentrifugetopelletcells.Decantsupernatant,leaving50-100µlofmediawithpellet.VortexthoroughlytoresUSPendcells.Add300µlTENS,vortexonhigh2-5s.Add150µl3MNaAcetatepH5.2,vortexonhigh2-5s.Spin4mintopelletcelldebrisandchromosomalDNA.Transfersupernatant(~450µl)toafreshtube(youcanusuallydothisbysimplypouringthesupernatant)andmixwellwith0.9ml95%EtOHwhichhasbeenprecooledto-20degC.NOTE:IfthestrainyouareworkingwithisnotendA-(e.g.MC1066,JF1754orTG1/lamBDa),itisrecommendedtodoa1:1phenol:chloroformextractionbeforeaddingtheethanol.Spin2minatroomtemperaturetopelletplasmidDNA.Discardsupernatant,andwashtwicewith70%EtOH(roomtemperature).Washingsimplyinvolvesgentlyaddingsome70%EtOH(usingasquirtbottle,forexample),andthenpouringitoff.Resuspendpelletin200µlTEpH8.0Use10µlperdigestandbesuretouseRNaseduringdigestion.
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