DNA纯化手册

PurificationofplasmidDNA(miniprep)withhighyieldsusingdiatomaceousearth

CorrespondencetoC.K.Pallaghy,e-mailC.Pallaghy@latrobe.edu.au

CopyrightK-S.KimandC.K.Pallaghy;1996,Allrightsreserved.Modified3/10/97

Introduction

Thischeapandsimplifiedprotocol,basedonHansenetal.,1995,giveshighyieldsofplasmidDNAaswellashighpurityandissuitableforcloning,PCR,sequencing,site-directedmutagenesisandinvitrotranscription,etc.Itisanextremelygoodmethodforroutineapplicationandprovidesagoodalternativewhenyieldstendtobelowduetolowplasmidcopynumberforanynumberofreasons(Sambrooketal.,1989).Yieldsareatleast3-5timeshigherthanthoseobtainedwithcommercialplasmidpurificationkitsandofteneventen-foldhigher.PlasmidDNAcanberecoveredfromdrainedliquidsusuallydiscardedwhenemployingcommercialkits.30-50%moreplasmidcanberecoveredifthesearepassedthroughadiatomaceousearthsystem.

Theyieldofplasmidusingtheprotocoldescribedhereishigherthanthatobtainedwithcommercialkitsevenifonlyusinglowornormalqualitydiatomaceousearth.Froma3mlofE.colicultureovernight,30-60microgofplasmid(e.g.pGEM-3Zf(+),Promega,Madison,WI,USA)canbeobtainedatapurityof1.8to2.0(260/280).Fora50mlovernightculture,500-800microgofplasmidcanbetypicallyobtained.

Themethodhasbeensuccessfullyemployedusingplasmidsrangingfromabout3.0to>100kb.Themethodbasicallyemploystwosteps-alkalineIysisofcells(Birnboim,1983)andelutionofDNAfromahome-madediatomaceousearthbindingmatrix(Hansenetal.,1995).WeusedaPromegaWizardTMminicolumn(Madison,USA),butotherbrands/typesofcolumnscanalsobeused.Centrifugationintheprotocoliscarriedoutat13,000rpm(oratleast>10,000rpm)onaminifugeunlessotherwisespecified.

Procedure

1.Growa3mlcultureofE.coliovernight(atleast16hrs)containinganappropriateantibiotic(e.g.ampicillin25-50microg/ml)

2.Harvestthecellsbycentrifugationfor2minandsuspendin300-500microlofSuspensionSolutionatroomtemperature.

3.Add300-500microlofLysisSolution,mixverygentlyandkeepatroomtemperatureforabout5min(butnomorethan5min)

4.Thenadd300-500microlofNeutralisingSolution.Invertgentlyseveraltimesandcentrifugeforatleast7min.Freshdiatomaceouscolumnsshouldbepreparedduringthistime(seeinstructionsforpreparationofcolumnsasmentionedlater).Althoughdiatomaceoussolutionsstorewell,thecolumnsdont.

5.CarefullytransferthesupernatantandmixwithapproximatelythesamevolumeofBindingBufferinasyringeandapplythemixturetothetopofafreshlymadediatomaceousearthcolumn(seeinstructionforpreparationofcolumnsasmentionedlater).Onceallthesolutionhasbeentransferred,andnosooner,applygentlesuctioninthesamemannerasdescribedforpreparationofthecolumn.

6.Add1mlofWashingSolutionandgentlydraininthesameway.Then,placethecolumnintoaneppendorftube^a)andcentrifugeforatleast3mintomakesurethatalltheWashingSolutioniscompletelyremovedfromthecolumn.Itisnecessarytorepeatstep6twice(toobtainhighqualityplasmids).

7.Placethewashedanddrainedcolumnintoaneweppendorftubeandadd50microlofpreheatedMQwater(70-80°C)orTEbuffer(atroomtemperature)toelutetheDNAandplaceatroomtemperaturefor10min(butnomorethan10min).

8.Centrifugethecolumnfor1-2min.Repeatingstep7-8twoorthreetimeselutesvirtuallyalloftheDNA.

a)Foreconomicpurposes,savetheemptiedeppendorftubefromstep4forstep6.

Solutionsrequired:

Allsolutionsshouldbepreparedinhighqualitydeionisedwater(MQ)suitableformolecularbiology.

1.SuspensionSolution

50mMTris-HCl,pH7.5-8.0,containing10mMEDTAand100microg/mlDNase-freeRNaseA.

Storeat4^oC.

However,inthecaseofplasmidisolatedfrombacteriasuchasXanthomonasspp.orPseudomonasspp.,producingexo-polysaccharideduringculture,useeitherSuspensionSolutioncontaining3%NaClorjust3%NaCl.Alternatively,approximately3%NaCl(finalconcentration)couldbeaddeddirectlytoabacterialculture.Mixthoroughlybeforeproceedingwithstep2.

2.LysisSolution

0.2MNaOHcontaining1%SDS

3.NeutralisingSolution

4Mpotassium-acetate,pH4.8

Place23.55gpotassiumacetateinmeasuringcylinderandfillto66mlmarkwithMQwater.Add28.5mlglacialaceticacid,mixandtitratewithabout1.5mlofconcentratedHCltopH4.8.Topupto100mlwithMQwater.

4.BindingBuffer

6Mguanidinehydrochloride

ItisnotnecessaryforguanidinehydrochloridetobedissolvedinTEbufferasdescribedinHansenetal.(1995)asMQwaterisequallygood.5Mor4Mworkswellbut6Mispreferable.Anythinglessthan3Mgivespoorresults.

5.WashingSolution

80%isopropanol(diluteto80%withMQwater).Ethanolisgenerallygoodasawashingsolution,exceptthatisopropanolischeaper.

6.TEBuffer

10mMTris-HCl,pH8.5,containing1mMEDTA

7.DiatomaceousEarthSolution

Thepreparationofthissolutioniscrucial.Suspendthediatomaceousearth(SigmaD-5384orotherbrands)at50mg/mlinwaterandleavetosedimentformorethan3hrs.Carefullydiscardasmuchofthewatercontainingthewhitegelatinouscolloidalsuspensionaspossible,butleavethesedimentintact.Repeatatleast3times(themore,thebetter).Iffinegelatinousmatterisfoundduringuse,thendiscardthesupernatantcarefullyandreplaceitwiththesameamountofwatertomaintainthesameconcentrationofdiatomaceousearthasabove.Again,anymilkysuspensionofdiatomaceousearthshouldberemovedasabove.Evennormalorlowquality(butacid-washed)diatomaceousearthgivesmuchbetteryieldsthananyofthecommercialkitstried.Highqualitydiatomaceousearthisonlynecessarywhenanultrapureplasmidpreparationisrequired.Wehavenottestedthedifferencebetweenahighlypureplasrnidandanultrapureplasmidpreparation,butwethinkthattheresultswillbethesameaslongastheplasmidpurityisbetween1.8to2.0(OD260/280)

Preparationofthediatomaceousearthcolumn

Thediatomaceousearthsolutionshouldberesuspendedthoroughlybeforeuse.

1.Placea2-5mlsyringetoaminicolumnandattachtoavacuumfitting,butnotapplyvacuumasyet!

2.Loadabout500-600microl(25-30mg)ofdiatomaceousearthsolutionontothecolumnandapplysuction.Onceallthesolutionhasbeenapplied,watchthecolumnfromaboveandbegintoapplygentlesuction.Disconnectthevacuumimmediatelywhentheliquidphasedisappearsandthesurfacebecomessolid.Thecolumnshouldlookgreyishwhite,withathinbrilliantwhitebandatthebottom.Ifthecolumnisbrilliantwhiteallthewayup,thevacuumhasbeenappliedfortoolong.DriedcolumnsdontbindDNA.

Ifyoudonothaveavacuumdeviceorsuitablesetup,connectasyringetothetopofthecolumnviatheluerlockandapplypressuregentlytoobtainthesameeffect.Besuretodisconnectthesyringefromthecolumnbeforepullingbackontheplunger.Thecolumnisnowreadytobeusedinstep5oftheproceduresection.ThesyringecanbereusedaftercleaningwithMQwaterordistilledwater.Thecolumncanalsobereusedafterappropriatecleaningasdescribedbelow.

1)Removethediatomaceousearthcompletelyfromthecolumn.

2)Soakthecolumnin0.1MHClforatleast1handboilfor10-20min.

3)WashitthoroughlyusingMQwaterordistilledwaterandautoclave.

4)Fitafilterinthecolumnusingayellowmicropipettipbeforeuse.

Keypointstoobserve:

a.UseaendA1^-E.colistrainforplasmidpropagationandisolationwheneverpossible.TheinstabilityofplasmidsisolatedfromendAl⁺bacterialstrainshasbeenreported(Schoenfieldetal.,1995).

b.Donotvortex,shakeorincubateformorethan5mininstep3.ThismaycauseshearingofgenomicDNAand/orlinerization(orunravelling)ofthesupercoiledplasmid.AIysistimeoflessthan5minisimportanttocausemaximumreleaseofplasmidwhileminimisingplasmiddenaturation.Thelysateshouldbeclearandviscous.

c.Useofcoldroomorlessthan7mincentrifugationmaygiverisetoadirtysupernatantinstep4.Ifforwhateverreasonthecentrifugationhastobeperformedatlowtemperature,themixtureshouldbetransferredtoroomtemperatureasquicklyaspossibleaftercentrifugation.

d.Inearlierprotocolsandinprotocolsofcommercialminiprepplasmidpurificationkits,lessthan1mincentrifugationisrecommendedtoremoveethanolfromeitherthebindingresinoradiatomaceousearthcolumn,butwefoundthatundertheseconditionssomeethanolstillremainedinthediatomaceousearth.Therefore,centrifugationshouldbeatleast3mininstep7.Ifnecessary,repeatthecentrifugationtwice.DNAwillnotbelost.

e.Useonlyhalfofthefirstvolumeduringstep9.If100microlisusedforthefirstelution,thenwerecommendlessthan50microlforthesecondelution.Ifthediatomaceousearthisfoundinthebottomofthetubefollowingcentrifugation,transferthesupernatantcarefullyintoaneweppendorftube.

TroubleshootingandHints

(i)Verylowyieldsofplasmid-thisisusuallyattributedtoalooselyfittingfilterinthecolumn.Checkwhetherthefilterinthecolumnisfittedlightly.Checktheplasmidcopynumber.Wasantibioticaddedornot?

(ii)LowpurityofplasmidwithanOD260/280,greaterorlessthan1.8-2.0.Thisusuallyarisesfromwhitegelatinousmatterremainingabovethediatomaceousearthwhenpreparingthesolution.Checkthediatomaceousearthsolution.CheckwhetherendA1^-/+cellswereused.

(iii)Vacuumisbestappliedfromasteadysourcesuchashousevacuum.Syringestendtostickandgiveburstsofvacuum.

(iv)Ifthesupernatantinstep5containscelldebrisinsuspensionbecauseofcarelesstransfer,thecolumnwillclog.Inthiscasedonotdiscardsample,butscratchcolumnsurfaceslightlywithpipettetiptounclogcolumn.

(v)CheapICNPracticalGradeguanidinehydrochlorideisquitesuitable,aslongasundissolvedsolidsareremovedbyfiltrationoncethetheoretically6Msolutionhasbeenmadeup.

(vi)HomemadefiltercolumnscanbemadeusingmicrocentrifugetubesasdescribedbyHansenetal.(1995),butwerecommendpiercingthebottomofthetubewithaneedle,fromtheinside,ratherthansnippingthebottomoff.

(vii)Inprincipleitshouldbepossibletoscalethisuptoamacro-prep.Wehaveonlyworkedwith20mlculturesperprep.

References:

1.Birnboim,H.C.1983.ArapidalkalineextractionmethodfortheisolationofplasmidDNA.MethodsEnzymol.100,243-255.

2.Hansen,NilsJakobV.,P.Kristensen,J.Lykke,K.K.MortensenandB.F.C.Clark.1995.Afast,economicalandefficientmethodforDNApurificationbyuseofahomemadebeadscolumn.BiochemistryandMolecularBiologyIntemational35(3),461-465.

3.SambrookJ.etal.,1989.MolecularCloning;ALaboratoryManual,2nded.,ColdSpringHarborLaboratoryPress,ColdSpringHarbor.

4.Schoenfeld,T.,J.Mendez,D.R.Storts,E.Portman,B.Patterson,J.FrederiksenandC.Smith.1995.EffectsofbacterialstrainscarryingtheendAIgenotypeonDNAqualityisolatedwithWizardPlasmidPurificationSystem.PromegaNotes,53,12-22.

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Triton-PrepMethodforbacterialDNAPurification

1. Grow5(largescale-15mlculture).Harvestinsingleeppendorftube(or15mldisposabletube).

2. Resuspendpelletwith300ulSTETbuffer(900ul).Afterresuspendingadd30ulRNase/lysozymemixture(100ul).

3. Boilforoneminute15seconds(oneminute45seconds).

4. Spininmicrofugeforatleast15minutes.

5. Takesupernatantandphenolextractwith150ul(500ul)STET-saturatedphenol.

6. Spinandtakesupernatant.Add1/10volume4Mlithiumchloride(autoclaved).Letsitonicefor5-10minutes.

7. Spinandtakesupernatant.Addequalvolumeisopropanol.RTfor5minutes.

8. Spin.Nopelletwillbevisible.Dontpanic,DNAisstucktosideallthewayuptube.

9. Important:Washwith80%ethanol(95%willcausetheresidualTritontoprecipitate)

10. Resuspendpelletin50-200ul.

    Lysozyme/RNasemixture

    10mg/mllysozyme

    1mg/mlRNase(usecheapgrade(BMB)ratherthanRNaseA,whichistooexpensive)

    50mMTris-HClpH8.0

    Storeat-20oCinsmallaliquots.Donotrefreezeafterthawing.

    STET

    8%sucrose

    5%TritonX-100

    50mMTris-HCl(pH8.0)

    50mMEDTApH8.0

    Filtersterilize.Storeat4oC

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    BacterialGenomicDNAPurificationviaQiagencolumns

    CulturesshouldbegrowninLBforbestresults.ToavoidoverloadingtheQiagencolumns,thecorrectnumberofcells/volumeofcultureshouldbedetermined.Qiagenrecommendsnotmorethan4x10E9cellsforamini-column,2x10E10forthemidi,and8x10E10forthemaxi.Forthisis~0.4-0.8ml,2.0-4.0ml,and10.0to20.0mlforovernightculturesinLB.

    1.Preparebuffersaccordingtorecipeatend.Equilibratealltoroomtemp.beforeuse.

    Amount/prepMiniMidiMaxiB11ml3.5ml11mlB20.35ml1.2ml4mlQBT2ml4ml10mlQC3ml15ml30mlQF2ml5ml15mlRNaseA0.2mg0.7mg2.2mglysozyme2mg8mg30mgQiagenprotease*0.9mg2mg10mg*orproteinaseK

    2.DissolveRnaseAinbufferB1toconcentrationof200ug/ml.StocksolutionsoflysozymeandproteinaseKcanbemadeindH2Otoconcentrationsof100mg/mland20mg/mlrespectively.

    3.Pelletcellsbycentrifugationat3000-5000xgfor5-10min.Removesupernatant.

    4.Resuspendthebacterialpelletin1/3.5/11ml(mini/midi/maxicolumns)ofBufferB1byvortexingattopspeed.

    5.Add20/80/300uloflysozymestocksolutionand45/100/500ulofproteinaseK.Incubateat37Cforatleast30min.

    6.Add0.35/1.2/4mlofBufferB2andmixbyinversionseveraltimes.Incubateat50Cfor30min.Mixwell;thisstepisimportantforefficientdeproteinization.Itisalsoimportantthatthelysatebecomesclearatthisstage.

    7.EquilibrateQiagengenomictipwith2x1/4/10mlBufferQBT,andallowthetiptoemptybygravityflow.

    8.Vortexthelysatefor5-10secandapplyontotheequilibratedcolumn.Again,allowittopassthroughcolumnbygravityflow.Flowcanbeassistedwithgentlepositivepressure(aplungerfroma10mlsyringefitsthemidicolumns)butitisalsoOKtodilutethelysatewithanequalvolumeofBufferQBTpriortoloading.Thelatterispreferablebyvirtueofpersonalexperience.

    9.Washthecolumnwith3x1ml/2x7.5ml/2x15mlofBufferQC.Allowbuffertopassthroughbygravityflow.Twowashesshouldbeenough.

    10.ElutethegenomicDNAwith2x1ml/5ml/15mlofBufferQF.Precipitatewith0.7volumes(1.4/3.5/10.5ml)isopropanol,equilibratedtoroomtemperature.TheDNAshouldbespoolable;ifnot,pellettheprecipitatebycentrifugationat5000+xg.Washtheprecipitatewith70%EtOHanddry.Resuspendintheappropriatesolvent(TE,water).

    Solutions:BufferComposition(Storage)B150mMEDTA,50mMTris/HCl,0.5%Tween20,0.5%TritonX-100(room[4CafteradditionofRNase])B23MGuHCl,20%Tween20(roomtemp)QBT750mMNaCl,50mMMOPS,15%ethanol,0.15%tritonX-100,pH7.0(roomtemp.)QC1.0MNaCl,50mMMOPS,15%ethanol,pH7.0(roomtemp.)QF1.25MNaCl,50mMTris/HCl,15%ethanol,pH8.5(roomtemp.)

    ChromosomalDNAExtractionfromGram-positiveBacteriaThisprocedurewasoriginallydevelopedforListeriamonocytogenesbuthasworkedwellwithotherGram+bacteriawevetried.

    Pelletcellsfrom10mlovernightculturesinBHIorLBandwashin5mlof0.1XSSC.Resuspendin1ml10mMTris-HCl(pH8.0)containing20%sucrose(v/v),addlysozymeto2.5mg/ml,andincubateat37Cfor45min.Add9mllysisbuffer(10mMTris-HCl[pH8.0],1mMEDTA,500mgpronaseB,1%SDS),andincubateadditional30minat37C.Phenolandchloroformextractlysedcells,andethanolprecipitatetheDNAwith0.1vol.3Msodiumacetate,pH4.8and2vol.95%ethanol.SpooloutDNAwithaglassrod,washoncewith80%ethanolbeforedrying.Somebacterialspeciesmayrequirealongerincubationinlysozyme.ForRenibacteriumsalmoninarum(aG+salmonpathogenweworkwith),lysozymeincubationsovernightat37CworkedverywellwithhighyieldsofDNA

    ref:Flamm,R.K.,Hinrichs,D.J.,andThomashow,M.F.1984.IntroductionofpAMb1intoListeriamonocytogenesbyconjugationandhomologybetweennativeL.monocytogenesplasmids.Infect.Immun.44:157-161.REFERENCEFULLTEXT:点击浏览该文件