DNAultrapurification

ThesuccessofDNApurificationcanhaveprofoundconsequencesonthesuccessorfailureofanyinjectioneffort.ThissummaryoftechniquesfromseveraldifferentsourceswascompiledbyBradPrestonattheUniversityofUtah.

PURIFICATIONOFDNAFORMICROINJECTION

(MethodsinEnzymologyp.775;ManipulatingtheMouseEmbryop.159)

IsolationofCodingElementsbyAgaroseGelElectrophoresis

1)Restrictyourcloneusingrestrictionsitesthatmaximizeseparationofthecodingelements(transgene+desired5"and3"sequences)fromvectorsequences(Note:somevectorsequencesappearlethaltoembryos).2)Electrophoresesampleofrestrictedmaterialon0.8%agarosetocheckcuttingefficiencyalongwithanappropriateMarkerandanuncutsampleofDNAinquestion.3)Purifyrestrictedsamplebyoneofthetwomethods(AorB)below:A)Qiagen-gelpurificationkit

1)ElectrophoreserestrictedDNAon0.8%agarose2)cutoutappropriatebands3)FollowQiagenkitinstructions

B)LowMeltingPointAgarosePurification

1)Make2%lowmeltingpoint(LMP)agarosegelinTAE+Ethidiumbromide.Allowgeltosolidifyat5^oCandstoreat5^oCuntiluse.2)Electrophoreseslowlyat5^oC(slowandcoldtoavoidmeltingofLMP).3)ExtractDNAfromGelase("FastProtocol"accordingtoGelasemanufacturer)

a)CutoutthedesiredbandoftheLMP-agaroseandtrimexcessagarose--UselongwavelengthUVlamptominimizedamagetoethidiumbromide-stainednucleicacids.b)Weighthegelsliceinataredtubec)Add1ul50xGelasebufferper50mgofgel(1ulofmoltenagarose=1mgofgel)d)Meltthegelslicecompletelyat70^oCe)Equilibratethemoltenagaroseat45^oCfor20minutes.f)AddtheappropriateamountofGelase(1unit/300mgof1%LMP-agaroseinTAE)andincubateat5^oCforatleastonehour--mayincubateovernight.

4)EthanolPrecipitationofNucleicAcidsfromGelase

a)Addonevolumeof5Mammoniumacetate.b)Add4volumesofroomtemperature100%ETOH;gentlymixc)Centrifugeat12,000gatroomtemperaturefor30minutes.DONOTUSEACENTRIFUGETHATISCOLD.d)Removesupernatant.e)Add3ml70%ETOH,gentlyswirlandcentrifugeat12,000gfor30minutesf)RemoveETOHanddrythepelletg)ResUSPendin1mlTEbuffer

5)ExtractpurifiedDNAwithPhenol/Chloroform(ManiatisE.3)

a)Addonevolumephenol(pre-equilibratedwithTrisbuffer,pH8.0)b)Centrifuge10minutes@10,000RPM,roomtemperature.c)CarefullyremoveaqueouslayerandaddequalvolumeofCHCl³:IsoamylAlcohol(24:1).d)Centrifuge10minutes@10,000RPM,roomtemperature.e)Removeaqueousphaseandextractwithwater-saturateddiethylether(ManiatisE.4).

6)FinalEthanolPrecipitation

a)Add1/10volume3MNaAcetateand2volumesofice-cold100%EtOH;gentlymixandincubateat-70^oCfor30minutes.b)Centrifugeat10,000RPMfor30minutesat4^oCc)Discardsupernatantd)Washpelletwithonevolumeice-cold70%EtOHe)Centrifugeat10,000RPMfor5minutesat4^oCf)Discardsupernatantandairdrypelletg)Resuspendin2.4mlTEbufferpH8.0

7)Cleanupgel-purifiedDNAbyoneofthefollowingmethods(detailedbelow):

*CsClcentrifugation*S&SElutipreg.minicolumns

CleanupofGel-PurifiedDNAbyS&SElutipreg.Method

 Low Salt Buffer High Salt Buffer 0.2 M NaCl 1.0 M NaCl 20 mM Tris*HCl 20 mM Tris*HCl 1.0 mM EDTA 1.0 mM EDTA pH 7.4 pH 7.4 20 ml 5 M NaCl 100 ml 5 M NaCl 10 ml 1 M Tris*HCl 10 ml 1 M Tris*HCl 1 ml 500 mM EDTA 1 ml 500 mM EDTA 400 ml dH20 300 ml dH20 pH to 7.4 pH to 7.4 Make up to 500 ml Make up to 500 ml Autoclave Autoclave 

Procedure

I.PreparationofMinicolumn

RemovetipprotectorontheElutip-dandcutoffthetipapproximatelyhalf-waybetweenthetipandfritsupportingthecolumnmatrix.Removetheprotectorcaponthetopoftheminicolumn.(Considerequilibrationwithlowsaltbufferfor2hourstoincreaserecovery.)LoadaLuer-locksyringewith5mloflowsaltbufferandattachtominicolumn.Washmatrixbypushingbufferthroughmatrixatapproximately0.5-1.0ml/min.

II.BindingofDNAtoMinicolumn

ReloadsyringewithDNAsamplewhichhasbeenresuspendedinlowsaltbuffer(5,10or20ml).AttachtheElutippre-filtertothecolumn.Attachsyringetopre-filterandcolumnandslowlypassallofthesamplethroughthefilterandminicolumn.Slowflowof1-2dropspersecondisnecessaryforefficientbindingoftheDNAtocolumn.

III.WashingtheColumn

Reloadthesyringewith2-3mllowsaltbuffer.Reconnectsyringetocolumnandpushlowsaltbufferthrough.Removethesyringeanddiscardthepre-filter.

IV.ElutionofDNAfromColumn

Loadthesyringewith0.4mlhighsaltbufferandreattachtothecolumn.ElutetheDNAfromtheminicolumnbypassingthehighsaltbufferthroughthecolumnintoa1.5mlmicrofugetube.Asecondelutionisoptional.PrecipitateDNAwith2volumesice-coldEtOHat-70^oCfor30min.PelletDNAinmicrofuge(12,000xgfor30min)anddiscardsupernatant.Rinsepelletwithcold(-20^oC)70%EtOHandrespinat12,000xgfor30min.Discardsupernatant,dryoffEtOHresidue,andresuspendpelletinfiltered(0.22u)injectionbuffer.QuantifyDNAbyOD260anddiluteinfiltered(0.22u)injectionbuffertoafinalconcentrationof1-2ngDNA/ul.

IMPORTANTNOTE!!THEINJECTIONBUFFERMUSTBEFREEOFPARTICULATEMATTERANDTHUSMUSTBEFILTEREDTHROUGHA0.22uMFILTERPRIORTOUSE.

CleanupofGel-PurifiedDNAbyCsClCentrifugation

1)Addexactly3gmsofCsCltothe2.4-mlsolutionofyourDNA.IMPORTANTNOTE!!YouarehighlyrecommendedtouseSuprapurereg.CsClfromMerck(catalog#2039)orequivalent.Impuritiesarelethaltosingle-cellmouseembryos.2)Mixthoroughlyandtransferto13x51mmpolyallomerultracentrifugetubesforSW50.1rotor3)Toptubeoffwithmineraloil(approximately2ml)4)Centrifugeat20^oC/45K/40hoursinSW50.1rotor5)Collectsmallfractionsofapproximately15drops/fraction6)Determinefraction(s)containingDNAofinterestbyelectrophoresison0.8%agarosegelbeingsuretoincludeanappropriatemarker.7)Pooldesiredfractionsanddialyze3timesagainst4litersofinjectionbuffer(10mMNaCl,5mMTrispH7.4,0.1mMEDTA).8)DetermineconcentrationofDNAviaopticaldensity.(Note:DNAconcentrationcanalsobeestimatedbyethidium-stainingintensityafteragarosegelelectrophoresisalongwithDNAstandardsofidenticallengthandknownconcentration.)9)Diluteinfilteredinjectionbuffertoobtainafinalconcentrationof1-2ngDNA/ul.