Largescaledouble-strandedDNAisolation

ThemethodusedfortheisolationoflargescalecosmidandplasmidDNAisanunpublishedmodification(16)ofanalkalinelysisprocedure(17,18)followedbyequilibriumultracentrifugationincesiumchloride-ethidiumbromidegrADIents(1).Briefly,cellscontainingthedesiredplasmidorcosmidareharvestedbycentrifugation,incubatedinalysozymebuffer,andtreatedwithalkalinedetergent.Detergentsolubilizedproteinsandmembranesareprecipitatedwithsodiumacetate,andthelysateisclearedfirstbyfiltrationofprecipitatethroughcheeseclothandthenbycentrifugation.TheDNA-containingsupernatantistransferredtoanewtube,andtheplasmidorcosmidDNAisprecipitatedbytheadditionofpolyethyleneglycolandcollectedbycentrifugation.TheDNApelletisresUSPendedinabuffercontainingcesiumchlorideandethidiumbromide,whichisloadedintopolyallomertubesandsubjectedtoultracentrifugationovernight.TheethidiumbromidestainedplasmidorcosmidDNAbands,equilibratedwithinthecesiumchloridedensitygradientafterultracentrifugation,arevisualizedunderlongwaveUVlightandthelowerbandisremovedwitha5ccsyringe.TheintercalatingethidiumbromideisseparatedfromtheDNAbyloadingthesolutionontoanequilibratedionexchangecolumn.TheA260containingfractionsarepooled,diluted,andethanolprecipitated,andthefinalDNApelletisresuspendedinbufferandassayedbyrestrictiondigestionasdetectedonagarosegelelectrophoresis.

Duringthecourseofthisworkseveralmodificationstotheaboveprotocolweremade.Forexample,initiallycellgrowthtimesincludedthreesuccessiveovernightincubations,beginningwiththeinitialinoculationof3mlofantibioticcontainingmediawiththeplasmidorcosmid-containingbacterialcolony,andthenincreasingtheculturevolumeto50ml,andthento4l.However,itwasobservedthatrecombinantcosmidDNAisolatedfromcellculturesgrownundertheseconditions,incontrasttorecombinantplasmidDNA,wascontaminatedwithdeletedcosmidDNAmolecules.However,thesedeletionsareavoidedbyperformingeachofthethreesuccessiveincubationsforeighthoursinsteadofovernight,althoughaslightyieldlossaccompaniedthereducedgrowthtimes.

Recently,adiatomaceousearth-based(19-22)methodwasusedtoisolatetheplasmidorcosmidDNAfromacelllysate.Thecellgrowth,lysis,andclearedlysatestepsareperformedasdescribedabove,butfollowingDNAprecipitationbypolyethyleneglycol,theDNApelletisresuspendedinRNasebufferandtreatedwithRNaseAandT1.NucleasetreatmentisnecessarytoremovetheRNAbydigestionsinceRNAcompeteswiththeDNAforbindingtothediatomaceousearth.AfterRNasetreatment,theDNAcontainingsupernatantisboundtodiatomaceousearthinachaotropicbufferofguanidinehydrochloridebyincubationatroomtemperature.TheDNA-associateddiatomaceousearththeniscollectedbycentrifugation,washedseveraltimeswithethanolbufferandacetone,dried,andthenresuspendedinbuffer.TheDNAiselutedduringincubationat65degC,andtheDNA-containingsupernatantiscollectedaftercentrifugationandseparationofthediatomaceousearthparticles.TheDNArecoveryismeasuredbytakingabsorbancereadingsat260nanometers.Afterconcentrationbyethanolprecipitation,theDNAisassayedbyrestrictiondigestion.

Protocol

1.PickacolonyofbacteriaharboringtheplasmidorcosmidDNAofinterestintoa12X75mmFalcontubecontaining2mlofLBmediasupplementedwiththeappropriateantibiotic(typicallyampicillinat100ug/ml)andincubateat37degC8-10hourswithshakingat250rpm.TransfertheculturetoanEhrlenmeyerflaskcontaining50mlofsimilarmedia,andincubatefurtherfor8-10hours.Transfer12.5mloftheculturetoeachof4litersofsimilarmedia,andincubateforanadditional8-10hours.

2.Harvestthecellsbycentrifugationat7000rpmfor20minutesin500mlbottlesintheRC5-BusingtheGS3rotor.Resuspendthecellpelletsinoldmediaandtransfertotwobottles,centrifugeasbefore,anddecantthemedia.Thecellpelletscanbefrozenat-70degCatthispoint.

3.Resuspendthecellpelletsinatotalof70mlofGET/Lysozymesolution(35mlforeachbottle)bygentlyteasingthepelletwithaspatulaandincubatefor10minutesatroomtemperature.(Note:DonotvortexthelysateatanytimebecausethismayshearthechromosomalDNA).

4.Addatotalof140mlofalkalinelysissolution(70mlforeachbottle),gentlymix,andincubatefor5minutesinanice-waterbath.

5.Add105mlof3MNaOAc,pH4.8(52.5mlforeachbottle),captightly,gentlymixbyinvertingthebottleafewtimes,andincubateinanice-waterbathfor30-60minutes.

6.ClearthelysateofprecipitatedSDS,proteins,membranes,andchromosomalDNAbypouringthroughadouble-layerofcheesecloth.Transferthelysateinto250mlcentrifugebottle,centrifugeat10,000rpmfor30minutesat4degCintheRC5-BusingtheGSArotor.

Forcesiumchloride-gradientpurification:

7a.Pooltheclearedsupernatantsintotoacleanbeaker,addone-fourthvolumeof50%PEG/0.5MNaCl,swirltomix,andincubateinanice-waterbathfor1-2hours.

8a.CollectthePEG-precipitatedDNAbycentrifugationin250mlbottlesat7000rpmfor20minutesat4degCintheRC5-BusingtheGSArotor.

9a.Dissolvethepelletsinacombinedtotalof32mlof100:10TEbuffer,5mlof5mg/mlethidiumbromide,and37gcesiumchloride(VarLacOidChemicalCo.,Inc.)(finalconcentrationofcesiumchlorideshouldbe1g/ml).

10a.Transferthesampleinto35mlpolyallomercentrifugetubes,removeairbubbles,sealwithrubberstoppers,andcrimpproperly.

11a.Centrifugeat60,000rpmto16-20hoursat15-20degCintheSorvallOTD-75Bultracentrifuge(DuPont)usingtheT-865rotor.

12a.VisualizetheethidiumbromidestainedDNAunderlong-waveUVlight,andremovethelowerDNAbandusinga5ccsyringeanda25gaugeneedle.(Itmaybehelpfulfirsttoremoveanddiscardtheupperband).

13a.Toremovetheethidiumbromide,loadtheDNAsampleontoanequilibrated1.5mlDowexcolumn,andcollect0.5mlfractions.EquilibratetheDowexAGresin(BioRad)bysuccessivecentrifugation,resuspension,anddecantingwith1MNaOH,water,andthen1MTris-HCl,pH7.6untiltheDowexsolutionhasapHof7.6.

14a.PoolfractionswithanA260of1.00orgreaterinto35mlCorexglasstubes,addonevolumeofddH2O,andethanolprecipitatebyadding2.5volumesofcold95%ethanol.Incubateatleast2hoursat-20degC,centrifugeat10,000rpmfor45minutesintheRC5-BusingtheSS-34rotor.Gentlydecantthesupernatant,add80%ethanol,centrifugeasbefore,decant,anddrytheDNApelletinavacuumoven.

15a.ResuspendtheDNAin10:0.1TEbuffer.

Fordiatomaceousearth-basedpurification:

7b.Poolthesupernatantsfromstep6into500mlbottlesandaddDNase-freeRNaseAandRNaseT1suchthatthefinalconcentrationofRNaseAis40ug/mlandRNaseT1is40U/ml.Incubateina37degCwaterbathfor30minutes.

8b.Addanequalvolumeofisopropanolandprecipitateatroomtemperaturefor5minutes.Centrifugeat9,000rpmfor30minutesintheRC5-BusingtheGS3rotor.DecantthesupernatantanddraintheDNApellet.

9b.ResuspendeachDNApelletin20ml10:1TEbuffer,andadd40mlofde-fineddiatomaceousearthinguanidine-HCl(100mg/ml)toeachbottle.AllowtheDNAtobindatroomtemperaturefor5minuteswithoccasionalmixing.Centrifugeat9,000for10minutesintheRC5-BusingtheGS3rotor.

10b.Decantthesupernatant,resuspendeachpelletin40mlofdiatomaceousearth-washbuffer,andcentrifugeasabove.

11b.Decantthesupernatant,resuspendeachpelletin40mlofacetone,andcentrifugeasabove.

12b.Decantthesupernatantanddrythepelletinavacuumoven.

13b.Resuspendthepelletin20mlof10:1TEbuffer,andelutetheboundDNAbyincubationat65degCfor10minuteswithintermittentmixing.

14b.Removethediatomaceousearthbycentrifugationat9,000rpmfor10minutesintheRC5-BusingtheGS3rotor.Repeatifnecessary.

15b.CombinetheDNA-containingsupernatantsandprecipitatetheDNAin35mlCorexglasstubesadding2.5volumesofcold95%ethanol/acetate.

16b.ResuspendthedriedDNApelletin2mlof10:0.1TEbufferandassayforconcentrationbyabsorbancereadingsat260nmorbyagarosegelelectrophoresis.