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,30-60"micro"gofplasmid(e.g.pGEM-3Zf(+),Promega,MADIson,WI,USA)canbeobtainedatapurityof1.8to2.0(260/280).Fora50mlovernightculture,500-800"micro"gofplasmidcanbetypicallyobtained.

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.ampicillin25-50"micro"g/ml)

2.Harvestthecellsbycentrifugationfor2minandsUSPendin300-500"micro"lofSuspensionSolutionatroomtemperature.

3.Add300-500"micro"lofLysisSolution,mixverygentlyandkeepatroomtemperatureforabout5min(butnomorethan5min)

4.Thenadd300-500"micro"lofNeutralisingSolution.Invertgentlyseveraltimesandcentrifugeforatleast7min.Freshdiatomaceouscolumnsshouldbepreparedduringthistime(seeinstructionsforpreparationofcolumnsasmentionedlater).Althoughdiatomaceoussolutionsstorewell,thecolumnsdon"t.

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

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

7.Placethewashedanddrainedcolumnintoaneweppendorftubeandadd50"micro"lofpreheatedMQwater(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,containing10mMEDTAand100"micro"g/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.Loadabout500-600"micro"l(25-30mg)ofdiatomaceousearthsolutionontothecolumnandapplysuction.Onceallthesolutionhasbeenapplied,watchthecolumnfromaboveandbegintoapplygentlesuction.Disconnectthevacuumimmediatelywhentheliquidphasedisappearsandthesurfacebecomessolid.Thecolumnshouldlookgreyishwhite,withathinbrilliantwhitebandatthebottom.Ifthecolumnisbrilliantwhiteallthewayup,thevacuumhasbeenappliedfortoolong.Driedcolumnsdon"tbindDNA.

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.If100"micro"lisusedforthefirstelution,thenwerecommendlessthan50"micro"lforthesecondelution.Ifthediatomaceousearthisfoundinthebottomofthetubefollowingcentrifugation,transferthesupernatantcarefullyintoaneweppendorftube.

TroubleshootingandHints

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

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

(iii)Vacuumisbestappliedfromasteadysourcesuchas"housevacuum".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.