ThismethodwasestablishedintheUniversityofMichiganTransgenicCoreforourusers.ThemethodisusedtopurifyBACDNAformicroinjectionintofertilizedmouseeggsandBACtransgenicmouseproduction..Thefollowingmethodyieldsclean,non-shearedDNA.SomeofinformationbelowwastakenfromTheNestGroupwebsite(TheNestGroup,Inc.TEL800-347-6378,FAX508-485-5736).

Materials:

ThekitsarenolongeravailablefromTheNestGroup,butcanbepurchasedfromClontech.

NucleoBondAX500Tip,ClontechCat.#4003-1Thisgivesthebestcompromiseofvolumemanagementandsize(capacity).Thiskitcontains10AX-500cartridges.

NucleoBondBufferSetI,ClontechCat.#4040-1.WithouttheBufferSetIyouwillhavetomakeupmoreoftheS1,S2,S3bufferssincethereisnotenoughinthePCkittodoublethevolumesofextractantsofanormalplasmid,whichisnecessarytodoBACs.

NucleoBondFoldedFilters,ClontechCat.#4062-1.Thefilterpaperswilleliminatethecentrifugationstepafterpptnofthecellulardebrisandwillclarifytheextractioninabout10minutesinsteadofa45minutespin.Thiswillreducetheexposuretimetonucleasesaswellasreducetheshearingpotentialofacentrifugation.Thisishighlyrecommended.

SpecialConditionsforPurifyingBACs:

WhenisolatingBACswiththeNucleobondAXalkalinelysis-basedprotocol,thepotentialalwaysexistsforlowerthanexpectedyieldstobeobtained.While,ingeneral,thereareanumberoffactorsthatcouldadverselyaffectplasmidyield,inthecaseoflowcopynumberplasmids,incompletebacteriallysisisthenumberoneculprit.Theprimaryreasonforthisisthatinordertogetthemaximumyieldforaparticularcartridgesize,manyresearchersgrowculturesoftheseplasmidsthatarelargerthancanbeefficientlyhandledbytheprescribedvolumesofbuffersforthecartridge.Thefirstandmostobvioussolutionistoincreasetheamountsofthebufferstobeusedinthesecasestoreduceviscosityandtopromotediffusion.

(A)SomeGoldenRulesforLysisBufferVolumes

Usethefollowingminimumvolumesofthecell-sUSPension/RNAsebufferS1,lysisbufferS2,andhighsaltco-precipitationbufferS3:Useaminimumof4.0mlofeachbufferper100mlofcultureregardlessofwhichcartridgesizeisbeingused.However,iftheparticularcartridgerequiresmorethanthis,thenusetheprescribedamountforthecartridge.Forexample,ifa100mlBACcultureistobeprocessedonanAX-500cartridgethenusetheprescribed12mlofS1,S2,andS3forthecartridge.Ontheotherhand,ifa500mlBACcultureistobeprocessedonanAX-500cartridgethenatleast20ml({500ml/100ml}*4)ofeachofthethreebuffersshouldbeusedaccordingtothisruletoinsurepropercelllysis.

(B)UseofanadditionalvolumeofN5ElutionBuffer

Iftheelutionstepisrepeatedoneadditionaltime(seeStep8,inAModifiedAlkalineLysisProcedureforthePurificationofPlasmidsandCosmids),upto30%moreDNAcanbeisolated.ThisisespeciallytruefortheAX-100andAX-500cartridges.NotetheflowratesofNucleobondAXcartridgesareuptotwotimesfasterthanforQiagenTipcartridgeswhichmeansthatcloggingfromhigherviscositysolutionsislesslikely(lesssensitivetocelldensityproblems),andtheamountoflossesfromendonucleaseswillbelowerduetoshortercontacttimes.

(C)UseofaDenaturingElutionBuffer

IthasbeenreportedinsomecasesthattheuseofahighGCcontenttypeofelutionbufferhelpstoincreasetheyieldsofverylargeplasmidssuchasBACsregardlessoftheirGCcontent.Theformulaforthiselutionbufferisasfollows:50%formamide,1.0MKCl,15%EtOH&0.1Tris-phosphate,pH8.5.Thisbuffershouldbeheatedto60CbeforeloADIng.FortherecommendedprocedureforpreparingthisbufferrefertoN5ElutionBufferPreparation.However,useofthisformamidebuffercanleadtocomplicationsintheprecipitationstepunlesscareistakentopreventsaltprecipitation(roomtemperaturepropanolprecipitationandcentrifugationisrequired),formamideremoval(asecondpropanolprecipitationisrequired)andpropanolremoval(anadditionalethanolprecipitationisrequired).

(D)NotesContributedbyChrisRussell,RESEARCHGENETICS,INC:crussell@resgen.com)

-SomeBACswillyieldbetterandmoreDNAwhengrownatroomtempto30Cratherthanat37C.Thisisnotgenerallytrue,butsomeBACsareunabletogrowvigorouslyat37C.

-ManyBACswillyieldbetterinLBbroththaninTerrificbrothorotherrichbroths.Itmaybeagrowthproblemoratechnicalproblemduethepresenceofahigherconcentrationofbacterialcells.

-ConcentrationmeasurementsbyUVspectrophotometryarenotveryreliableandmaynotbemeaningful,sinceyoucanhaveabsorbingcontaminantsaswellassignificantamountsofE.coliDNA.SameistrueforflourometryduetothepresenceofE.coliDNA.SamegoesforcheckingtheBAConaregularagarosegel.Bypulsed-fieldgelelectrophoresisofNotIdigestedBACDNAyoucanevaluatetheintegrity,quality,andquantityofBACDNA.

Method:

UsingthismodifiedmethodofBirnboimandDoly,bacterialcellsarelysedbyaNaOH/SDSsolution.ChromosomalandplasmidDNAarepartiallydenaturedunderthesealkalineconditions.Itisimportanttocontrolthedurationofthisdenaturingstepthoroughly.Thefollowingadditionofpotassiumacetateisacrucialstepaswell.ItresultsinaprecipitatecontainingthechromosomalDNAandothercellularcompounds.PlasmidDNAstaysinsolutionandispurifiedtohomogeneityonthecorrespondingNUCLEOBONDAXcartridge.Forbuffervolumesandoperationconditionsseethebelow.Pleasereadtheremarksbeforeyoustartyourpreparation.

Use100-500mlofbacterialculture(A600approximately1O.D.)inconjunctionwithoneAX-500cartridge.

Celldisruption:

1.CarefullyresuspendthebacterialcellpelletinbufferS1:12ml(seeNoteAabove)

2.AddtheappropriatevolumeofbufferS2:12ml(seeNoteAabove)Mixgentlybyinvertingthetube,andincubateatroomtemperatureor5min.DonotvortextopreventthereleaseofchromosomalDNAfromthecelldebris.

3.AddbufferS3:12ml(seeNoteAabove),Mixgentlybyinverting6-8timesuntilahomogeneoussuspensionisformed.Incubatethemixtureonicefor10minutestobegintheprecipitationofSDSandcellulardebris.Putthefoldedfilterontoa50mltubeorequilibratedNucleobondAXcartridge,wetitwith0.5-1.0mlofwaterandfillitwiththecooledlysate.Collecttheclearedflowthrough.ThesefiltersaresuitableforplasmidisolationwithNucleobondAX100andlargercartridges,wherethevolumeofthelysateissufficienttoeffectacompletehydrationandwashofthefilterpaper.TogetthemaximalrecoveryofDNA,rinsethefilterpaperwithanadditional1.5mlofwater.

Equilibration:4.EquilibrateanAX-500cartridgewith5mlbufferN2

Adsorption:5.LoadthecollecttheclearedflowthroughfromthefilterpaperontoanequilibratedanAX-500cartridge.

Wash:6.Washcartridgewith2x12mlbufferN3.

Elution:7.EluteDNAwith6mlbufferN5(seenotesBandCabove)

Ifthiselutionstepisrepeatedoneadditionaltime,upto30%moreDNAcanbeisolated.ThisisespeciallytruefortheAX-100andAX-500cartridges.

Precipitation:8.PrecipitateDNAbyadding0.7volumesofroomtemperatureiso-propanol.Centrifugeat>12000xgat4Cfor10-20minutes.Washthepelletwith70%ethanol,airdrybriefly(about5minutes),anddissolveinmicroinjectionbuffer.

ResuspendDNA9.ResuspendDNAin100ulmicroinjectionbufferavailablefromtheTransgenicCore.

Quantitation:10.Checkconcentrationby0D260/280.WeandothershaveobservedthatthereisnodifferenceinefficiencyofproducingtransgenicmicewithcircularBACDNAorlinearizedBACDNA.YoumaysubmiteitherformofDNAformicroinjection.Whenyoudoso,alsosubmit10microlitersofNotIdigestedBACDNAforpulsedfieldanalysis.ThiswillallowustoassessthepurityofyourDNApreparation.WewillverifytheconcentrationofyourconcentratedDNAsampleandadjustitformicroinjectionto0.5to1.0ngperul.

DNAStorage:StoreresuspendedDNAat4C.

BufferStorage

S1-50mMTris-HCl,10mMEDTA,100µgRNAseA/ml,pH8.0,4CS2-200mMNaOH,1%SDS,RTS3-2.8MK-Acetate,pH5.2,4CN2-100mMTris,15%ethanol&900mMKCladjustedwithphosphoricacidtopH6.3,RTN3-100mMTris,15%ethanol&1150mMKCladjustedwithphosphoricacidtopH6.3,RTN5-100mMTris,15%ethanol&1000mMKCladjustedwithphosphoricacidtopH8.5,RT

*Thissaltconcentrationissufficientfortheelutionstep,becauseoftheincreasedpHvalueofthisbuffer.

TheRNAse(alreadyheattreatedandDNAse-free),mustbeaddedtobufferS1beforeuse.S1shouldbestoredthenat4C.TheSDSinbufferS2willprecipitateattemperaturesbelow20C.Ifthisisthecase,storethebottleforafewminutesatabout30Cto40Candmixwellandequilibratetoroomtemperaturebeforeuse.TheSDSisremovedbybufferS3(whiteprecipitate)andwillnotbeloadedonthecartridge.Thisstepisveryimportant!Takecarethatthesupernatantofstep5isclear!SDSwillclogthecartridgeandpreventtheadsorptionofnucleicacids.