SpecialConsiderationsNevervortex,onlyrockslowlyorswirltomix.WhenredissolvingDNApellets,placetubesonaflatrotatorforabout15minutesatroomtemperature.

1.Inoculate2.5mlofLB(+antibiotic)withsinglecolonyordirectlyfromglycerolstock.,Letgrowovernightshakingat37°C.

2.Use1mlofovernightculturetoinoculate500mlofLB(+antibiotic)x2foratotalculturevolumeof1L,shakeovernight(about16hrs)at37°C.

3.Collectcellsbycentrifugationin250mlx4Nalgenebottles,(H-6000Arotor,3800rpm),10minutes,4°C.

4.WashcellsbyresUSPendingin25mlsofICECOLD10mMNaCl(0.1ml5Minto50mlfinal)per250mlbottle,pelletat3800rpmfor10minutesat4°C.

5.Decantsupernatant,(freezingthepelletatminus80°Covernightatthisstepincreasescelllysisefficiency),resuspendpelletfromeach250mlculturein10mlofSolutionIbypipetingupanddownwith10mlstripette:Letstandfor5minutesatroomtemperature.

SolutionI10mMEDTA,pH8.0for100mlbringup2mlof0.5MEDTA,pH8.0to100ml

6.Add20mlofSolutionIIpereach250mlofculture,swirltomix(DONOTINVERT)andletstandfor5minutesatroomtemperature.

SolutionII(makeupfreshEVERYtime)for50ml0.2MNaOH1mlof10NNaOH(20gNaOHinto50mlsfor10N)1%SDS5mlsof10%SDS(50ginto500mlsfor10%,filtersterilize)

7.Add15mloficecoldsolutionIIIpereach250mlofculture,mixgentlybyswirlingandplaceonicefor10minutes.(Note:Toinsureproperneutralization,youmaywanttokeeponiceforlongerthan10minutes.Clontech’sNucleobondAX-500protocolcallsfor30-40minutes.Wekeeponiceforminimally20minutes.)

SolutionIIIK-acetate(7.5MStock,formulaweight98.15,use184.03ginto250mlautoclavedMilliQH2O,storeatroomtemperature)50mlGlacialAceticAcid23mlWater127ml

8.Spinat15,300xgRCFMax(10,000rpmBeckmanJA14rotor;10,000rpmSorvallSLA-1500rotor)for15minutesat4°C,removesupernatant,thenrepeatspinonsupernatantinfreshbottle.(Note:YoumaywantfiltersupernatantthroughapleatedWhatman#1filterpaperusingautoclavedfunnels.)

9.Add45mlofisopropanoltothesupernatantandmix.Spinat3850xgRCFMax(5,000rpmBeckmanJA14rotor;5,000rpmSorvallSLA-1500rotor)for15minutes.

10.Pouroffthesupernatantanddissolvepelletin9mlof10T/50E(10mMTris-HCLpH8.0(0.5mlof1M);50mMEDTApH8.0(5mlof0.5M))thentransfertoanOakridgetube(akaSS34).Add4.5mlofK-acetate(7.5M),mixgently,andkeepatminus80°Cfor15-30minutes.

11.Thawcompletely(avoidmixing)andspinat3,000xgRCFMax(5,000rpmBeckmanJA20rotor;4,500rpminSorvallHB-6swingrotor)for10minutes.Combinesupernatantsintoa50mlconicaltube(Note:Youshouldhaveatotalvolumeof54mlsatthisstep.)Splitsupernatantbytransferring9mlsinto6akridgetubes.Add22.5mlof100%Ethanoltoeachtube,mixgently.

12.PelletDNAat3000xgRCFMax(5,000rpmBeckmanJA20rotor;4,500rpmSorvallHB-6rotor)for10minandredissolveprecipitatein1.3mlof50T/50E(50mMTris-HCLpH8.0(2.5ml1MTris-HCL);50mMEDTApH8.0(5.0ml0.5MEDTA)).Poolvolumesx3intoa2059Falcontube(Note:Youshouldhave22059Falcontubeswith4mlsineachatthisstep.)

13.Extractaqueouswith2mlofphenol(tris-buffersaturated)bygentlyrocking,letsitfor3minutes.Add2mlofCHCl3,mixbygentlyrocking,thenseparatephasesby3-5,000rpminHB-6swingrotor.

14.Removeaqueouslayer(~4ml)withwide-bore2mlstripette(breaktheendoffthe2mlplasticPipettetomakeitwide-bore)intoFalcon2059tube.Add1/10(400ul)volumeof3MNaOAcpH7.0.Add2.5volumesof100%Ethanol(~10ml),mixgentlybyinversionandplaceatminus20°Cforatleast30minutes,preferably2hours.

15.PellettheDNAbycentrifugationat6000rpminswingingbucketSorvallHB-6rotorat4°Cfor15minutes.

16.Dissolvepelletin2ml50T/25E(50mM(2.5mlof1M)Tris-HCLpH8.0,25mM(2.5mlof0.5M)EDTApH8.0).Oncedissolved,combinevolumesintoone2059Falcontube(totalof4ml).Add4.8gCsCl,dissolvebygentlerocking/warming(inyourhand).Whencompletelydissolved,add0.4mlEtBr.(fromthispointPROTECTFROMLIGHTtoavoidnickingoftheDNA)

17.Spinat3000rpminswingingbucketrotortoremoveprecipitatedprotein,poursupernatantintonewtubeavoidingproteinscum.Transfersupernatanttoultracentrifugetube.(Sorvall#03945,TubePA,6mlvolume)Density~1.6g/mlongrADIents.

18.Balancetubestowithin0.1g,mountinSorvallTV865rotor,tightencapsto20lbswiththetorquewrench.Ultracentrifugeovernightat55,000rpm,20°C,zonal4,NObrakeondeceleration..

19.Insert25-30gneedleintotopoftubetoprovidepressureoutlet,pullbandswith18gneedleand1mlsyringetopulltheBACband(SupercoiledBACDNAisthelowerband).RemovebandinthesmallestpossIBLevolume(shouldbe<1ml).

21.ExtractEtBrwithTEpH7.5/100mMNaCl-saturatedbutanol(1:1volumeratio),>5times,oratleast1extractionpaststepwhereallorangecolorisremoved.Note:tomakeTE/NaClsaturatedbutanol,makeup10mMTrispH7.5,1mMEDTA,100mMNaClthenaddtobutanolwithintermittentmixinguntiltwophasesareobserved(butanolislessdenseandwillbetheupperphase).

22.DialyzetheDNAusingthreechangesof1XPolyamineMicroinjectionbuffer(1litervolumeperchange)at4°Cspinningveryslowlyinbakedglasswaretoavoidnucleases.Wemakeupthepolyaminebuffer,sterilefilteritandthenuseitimmediatelyfordialysis.Placedialysismembraneintonewbeakerevery4hourswithfinalovernightdialysis.1XPolyaminebufferconsistsof:10mMTris,pH7.5,0.1mMEDTA,30microMspermine,70microMspermidine,100mMNaCl

For1liter:10mMTris7.5use1mlof1MTris-HCl,7.5stock(autoclaved)0.1mMEDTAuse20ulof0.5MEDTA,8.0(autoclaved)10mMNaCluse2mlof5MNaClstock(autoclaved)1XPolyAminesuse100ulof1000XPolyAmineStock(30mMSpermine,70mMSpermidine)MilliQH2Ouptofinalvolumeof1000mlandthenfiltersterilize

23.TakeanaliquotafterDialysisandevaluateconcentrationbyA260toobtainaroughestimateofyield.Atthispointconcentrationislowenoughthatit"sbesttoread100ulofdialysatedirectlyinthecuvetteandthendiscard.UseleftoverdialysisbufferforblankonA260.StoreBACDNAat4C.