Solutions

Procedure

•Starta500mlculturewiththeappropriateantibioticapproximately24hoursinadvancebyinnoculatingasinglecolony.

•Harvestthecellsbyspinningina500mlbottleintheJ6Bat4.2Kfor20minutes.ResUSPendthepelletgentlyin30mlSolutionIwith1mg/mllysozyme(Sigma#L6876).

•Transferimmediatelytoa250mlbottleoniceandadd60mlSolutionII,followedby45mlSolutionIII.ImmediatelyspinintheSorvallGSArotorat10Kfor10".

•Transferthesupernatanttoaclean250mlbottleandspinfor10"at10K(optionalifthereisstillsomeprecipitateafterthefirstspin).Transferthesupernatantfromthisspintoanother250mlbottlefilteringthroughseverallayersofcheesecloth.

•Add1volumeofisopropanolandincubateatroomtemperaturefor5-10minutesfollowedbya10Kspinfor30".

•Thoroughlydrythepelletandresuspendin6mlTE.Transfer5.5mltoa13mlsnaptoptubeandadd6.1gCsClplus0.3mlEthBr(10mg/ml).

•SpinintheBrinkmanSpeedfugefor10"at6KandtransferthesupernatanttoaQuickSealTube.Topoffwithheavymineraloil,balanceandseal.

•Spinat56Krpmforatleast20hours,removetheplasmidDNAbandwithaneedleandextractEthBrwithNaClsaturatedIsopropanol.Alternately,extractwithisoamyalcoholandprecipitatebyadding2volumesof70%EtOH.Wash,dryandresuspendin0.5mlTE.

•IftheEthBrwasremovedbyNaClsaturatedisopropanol,dialyzeagainst2litersofTEat4degreesfor24-48hours,andquantitate.

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