CsClPrepofPlasmidDNA Thisisastandardlargescaleprep.forplasmidDNAwhichgivesayieldof0.5-1.0mg.IhavemadesomeminorchangestotheMHBprotocol. Solutions SolutionI,II,IIIfromprotocolD.1. Tris/EDTApH7.5(optional) 20ml1MTris7.5 4ml0.5MEDTA8.0 upto2literswithQ storeat4degreesfordialysis DialysisTubing(optional) Thetubingisstoreddryat4degrees(Spectrapore12,000-14,000molecularweightcutoff) cuta1-2metersegmentandimmerseinQ autoclavefor10minutesonfastexhaust storeat4degreesin20%EtOH priortouse,washinTE 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.
