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Random subclone generation

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Randomsubclonegeneration

ThegenerationofDNAfragmentsbysonicationisperformedbyplacingamicrocentrifugetubecontainingthebufferedDNAsampleintoanice-waterbathinacup-hornsonicatorandsonicatingforavaryingnumberof10secondburstsusingmaximumoutputandcontinuouspower(10),essentiallyasdescribedbyBankierandBarrell(11).Duringsonication,temperatureincreasesresultinunevenfragmentdistributionpatterns,andforthatreason,thetemperatureofthebathismonitoredcarefullyduringsonication,andfreshice-waterisaddedwhennecessary.TheexactconditionsforsonicationaredeterminedforagivenDNAsamplebeforeapreparativesonicationisperformed.Approximately100ugofDNAsample,in350ulofbuffer,isdistributedintotenaliquotsof35ul,fiveofwhicharesubjectedtosonicationforincreasingnumbersof10secondbursts.Aliquotsfromeachtimepointareelectrophoresedonanagarosegelversusthephi-X174sizeMarker(12)todeterminetheapproximateDNAfragmentsizerangeforeachsonicationtimepoint.Onceoptimalsonicationconditionsaredetermined,theremainingfiveDNAaliquots(approximately50ug)aresonicatedaccordingtothosepre-determinedconditions.Aftersonication,thefivetubesareplacedinanice-waterbathuntilfragmentend-repairandsizeselection,discussedbelow.

Protocol

1.PreparethefollowingDNAdilution,andaliquot35ulintoten1.5mlmicrocentrifugetubes:

DNA100ug10XTMbuffer35ulsterileddH2Oq.s.FinalVolume350ul

2.Todeterminetheoptimalsonicationconditions,sonicatetheDNAsamplesinfiveofthetubesinaHeatSystemsUltrasonicsW-375cuphornsonicatorseton"HOLD","CONTINUOUS",andmaximum"OUTPUTCONTROL"=10underthefollowingconditions:
TubeNo.10secondbursts1122334455
WehaverecentlylearnedthattheGenomeCenteratWashingtonUniversityandtheSangerCentersettheOUTPUTCONTROLtothelowestpossIBLesettings.BecauseatpresentweusetheNEBulizer(seethenextsectionbelow),wehavenotinvestigatedthisfurther.
2.CooltheDNAsamplesbyplacingthetubesinanice-waterbathforatleast1minutebetweeneach10secondburst.Replacetheice-waterbathinthecuphornsonicatorbetweeneachsample.
3.Centrifugethesamplestoreclaimcondensationandelectrophoresea10ulaliquotfromeachsonicatedDNAsampleonaagarosegelversusthephi-X174/HaeIIIsizemarker(Pharmacia15611-015).
4.Basedonthefragmentsizerangesdetectedfromagarosegelelectrophoresis,sonicatetheremaining5tubesaccordingtotheoptimalconditionsandthenplacethetubesinaice-waterbath.
YoucanpurchaseNebulizer,Number4101or4101UO,fromalocalsupplier,whosenameyoucanobtainbycallingthemanufacturer:
IPIMedicalProductsInc.3217NorthKilpatrickChicago,IL60641phone:(773)777-0900
ThepresidentofIPIisWalterLevinesoifyouhaveanytroublesorderingthembesuretoaskforhimand/ortotellthemthatthesedevicesare:"NOTINTENDEDFORPATIENTUSE"
BasicallywefollowaprotocolsenttousbySteveSurzyckiattheDepartmentofBIOLOGy,IndianaUniversity.
Therearetwosmallproblemsthatwesolvedasfollows:
1.Youhavetocovertheholewherenormallythemouthpiecegetsattachedto;coverthatholewithacapQS-TfromISOLABInc.(Drawer4350Akron,OH44303,100capsfor$9.50).
2.Theotherproblemthatmayoccuristhatthenebulizerleakswherethehoseforthenitrogengetsattached.ItseemsthatNalgenetubing(VIgrade3/16"ID)sealsbetterthatthetubingwhichcomeswiththenebulizer.Thenebulizermightstillleaksomewhatatthetop,youcan"tavoidthat.
NebulizerSummary:
Anebulizercontaining2mlofabufferedDNAsolution(approximately50ug)containing25-50%glycerolisplacedinanice-waterbathandsubjectedtonitrogengasatapressureof8-10psifor2.5minutesfornebulizingBACs(10,13).NitrogengaspressureistheprimarydeterminantofDNAfragmentsize,andalthoughpressurestudiesshouldbeperformedwitheachBAC,cosmidorplasmid,apressureof8-10psialmostalwaysresultedinthedesired(1kb-4kbp)fragmentsizerange.Asdiscussedaboveforsonication,theuseofanice-waterbathfornebulizationalsoiscriticaltothegenerationofevenlydistributedDNAfragments.Duringthenebulizationprocess,unavoidableleaksareminimizedbysecurelytighteningthelidfornebulizerchamberandsealingthelargerholeinthe
toppiecewithaplasticcap.Toprepareforfragmentend-repair,thenebulizedDNAtypicallyisdividedintofourtubesandconcentratedbyethanolprecipitation.
Protocol
1.Modifyanebulizer(IPIMedicalProducts,Inc.4207)byremovingtheplasticcylinderdripring,cuttingofftheouterrimofthecylinder,invertingitandplacingitbackintothenebulizer.Sealthelargeholeinthetopcover(wherethemouthpiecewasattached)withaplasticstopperandconnecta1/4inchidlengthofTygontubing(whicheventuallyshouldbeconnectedtoacompressedairsource)tothesmallerhole.
2.PreparethefollowingDNAsampleandplaceinthenebulizercup:
DNA50ug10XTMbuffer200ulsterileglycerol0.5-1mlsterileddH2Oq.s.2ml
3.Nebulizeinanice-waterbathat30psifor2.5minutesforplasmid,or8-10psifor2.5minutesforBACs,PACs,fosmidsorcosmids.
4.Brieflycentrifugeat2500rpmtocollectthesamplebyplacingtheentireunitintherotorbucketofatabletopcentrifuge(BeckmanGPRtabletopcentrifuge)fittedwithpiecesofstyrofoamtocushiontheplasticnebulizer.
5.Distributethesampleintofour1.5mlmicrocentrifugetubesandethanolprecipitate.ResUSPendthedriedDNApelletin35ulof1XTMbufferpriortoproceedingwithfragmentend-repair.
SincebothsonicatedandnebulizedDNAfragmentsusuallycontainsingle-strandedends,thesamplesareend-repairedpriortoligationintoblunt-endedvectors(10,11).AcombinationofT4DNApolymeraseandKlenowDNApolymeraseareusedto"fill-in"theDNAfragmentsbycatalyzingthe3"-5"incorporationofcomplementarynucleotidesintoresultantdouble-strandedfragmentswitha5"overhang.Additionally,thesingle-stranded3"-5"exonucleaseactivityofT4DNApolymeraseisusedtodegrade3"overhangs.Thereactionsincludedthetwoenzymes,buffer,anddeoxynucleotidesandareincubatedat37degC.
Followingfragmentend-repair,theDNAsamplesareelectrophoresedonapreparativelow-meltingtemperatureagarosegelversusthephi-X174marker,andafterappropriateseparation,thefragmentsinthesizerangefrom1-2Kbpand2-4Kbpareexcisedandelutedseparatelyfromthegel,asdiscussedabove.Alternatively,thefragmentscanbepurifiedbyfractionationonaSephacrylS-500spincolumnasalsodiscussedabove.Inbothinstances,thepurifiedfragmentsareconcentratedbyethanolprecipitationfollowedbyresuspensioninkinasebuffer,andphosphorylationusingT4polynucleotidekinaseandrATP.ThepolynucleotidekinaseisremovedbyphenolextractionandtheDNAfragmentsareconcentratedbyethanolprecipitation,dried,resuspendedinbuffer,andligatedintoblunt-endedcloningvectors.ItshouldbenotedthatbecauseasignificantportionofnebulizedDNAfragmentsareeasilyclonedwithoutend-repairorkinasetreatment,thesetwostepscanbecombinedwithoutsignificantlyaffectingtheoverallnumberofresultingtransformedclones(seesectionV.B.onpurificationofPCRfragmentsforcloning,whichdescribesamethodforsimultaneousend-repairandkinasetreatment).
Protocol
1.Toeachtubecontaining35ulofDNAfragments(fiveofsonicatedDNAandfourofnebulizedDNA),add:
0.25mMdNTPs2ulT4DNApolymerase3ul(3U/ul)KlenowDNApolymerase2ul(5U/ul)42ul
T4(203L)andKlenow(210L)DNApolymerasesfromNewEnglandBiolabs.
2.Incubateatroomtemperaturefor30minutes.
3a.Add5ulofagarosegelloADIngdyeandapplytoseparatewellofa1%lowgeltemperatureagarosegelandelectrophoresefor30-60minutesat100-120mA.
4a.ElutetheDNAfromeachsamplelane,ethanolprecipitate,andresuspendthedriedDNAin36ulofsterileddH2Oandadd4ulof10Xdenaturingbuffer.Thereshouldbefivetubesforsonicatedfragmentsandfourtubesfornebulizedfragments.
5a.Incubateat70degCfor10minutes,andplacethesamplesinanice-waterbath.
6a.Addthefollowingreagentsforthekinasereactionandincubateat37degCfor10-30minutes:
10mMrATP1ul10Xkinasebuffer5ulT4polynucleotidekinase1ul(30U/ul)FinalVolume47ul
T4polynucleotidekinase(70031)fromUnitedStatesBiochemicals.
7a.Poolthekinasereactions,phenolextract,ethanolprecipitate,andresuspendthedriedDNAfragmentsin40ulof10:0.1TEbuffer.Thisyieldsatypicalconcentrationof500-1000ng/ul.
Alternativelytheend-repairandphosphorylationstepscanbecombined:
1b.ResuspendDNAin27ulof1XTMbuffer.Addthefollowing:
10Xkinasebuffer5ul10mMrATP5ul0.25mMdNTPs7ulT4polynucleotidekinase1ul(3U/ul)KlenowDNApolymerase2ul(5U/ul)T4DNApolymerase3ul(3U/ul)------------------------------------------------------FinalVolume50ulnote:iftheDNAhasbeenshearedbynebulizing,theT4DNApolymeraseadditionheremaynotbenecessary.
2b.Incubateat37degCfor30minutes
3b.Add5ulofagarosegelloadingdyeandapplytoseparatewellofa1%lowmeltingtemperatureagarosegelandelectrophoresefor30-60minutesat100-120mA.
4b.ElutetheDNAfromeachsamplelane,ethanolprecipitate,resuspendin10ulof10:0.1TEbuffer.
DNAligationsareperformedbyincubatingDNAfragmentswithappropriatelylinearizedcloningvectorinthepresenceofbuffer,rATP,andT4DNAligase(10,11).Forrandomshotguncloning,sonicatedornebulizedfragmentsareligatedtoeitherSmaIlinearized,dephosphorylateddouble-strandedM13replicativeformorpUCvectorbyincubationat4degCovernight.ApracticalrangeofconcentrationsisdeterminedbasedontheamountofinitialDNA,andseveraldifferentligations,eachwithanamountofinsertDNAwithinthatrange,areusedtodeterminetheappropriateinserttovectorratiofortheligationreaction.Inaddition,severalcontrolligationsareperformedtotesttheefficiencyoftheblunt-endingprocess,theligationreaction,andthequalityofthevector(10,11).TheseusuallyincludedparallelligationsintheabsenceofinsertDNAtodeterminethebackgroundclonesarisingfromself-ligationofinefficientlyphosphatasedvector.Parallelligationsalsoareperformedwithaknownblunt-endedinsertorinsertlibrary,typicallyanAluIdigestofacosmid,toinsurethattheblunt-endedligationreactionwouldyieldsufficientinsertcontainingclones,independentoftherepairprocess.
Protocol
1.Combinethefollowingreagentsinamicrocentrifugetube,andincubateovernightat4degC:
DNAfragments100-1000ngcloningvector2ul(10ng/ul)10Xligationbuffer1ulT4DNAligase(NEB202L)1ul(400U/ul)sterileddH2Oq.s.10ul
ThecloningvectortypicallyisSmaI-linearized,CIAP-dephosphorylatedpUCvector(Pharmacia27-4860-01)asseveralyearsagoweswitchedfromM13topUC-basedshotguncloning.Theadvantageofobtainingtwosequencereadsoffoneisolatedshotgunsub-cloneseemstooutweighthedisadvantageofafewbaseslessindouble-strandedvssingle-strandedreadlengths.Insomeinstances,including5%PEGintheligationreactionsalsoseemstoslightlyimprovetheligationefficiency.
2.IncludecontrolligationreactionswithnoinsertDNAandwithaknownblunt-endedinsert(suchasAluIdigestedcosmid).
Therearetwomainmethodsforpreparationofcompetentbacterialcells(14)fortransformation,thecalciumchlorideandtheelectroporationmethod.Forthecalciumchloridemethod,aglycerolcellculturestockoftherespectiveE.colistrainisthawedandaddedto50mlofliquidmedia.Thisculturethenispreincubatedat37degCfor1hour,transferredtoanincubator-shaker,andisincubatedfurtherfor2-3hours.Thecellsarepelletedbycentrifugation,resuspendedincalciumchloridesolution,andincubatedinanice-waterbath.Afteranothercentrifugationstep,theresultingcellpelletagainisresuspendedincalciumchloridetoyieldthefinalcompetentcellsuspension.Competentcellsarestoredat4degC,foruptoseveraldays.
CalciumChlorideProtocol
1.ThawafrozenglycerolstockoftheappropriatestrainofE.coli,addittoanErlenmeyerflaskcontaining50mlofpre-warmed2xTY(1)media,andpre-incubateina37degCwaterbathfor1hourwithnoshaking.Furtherincubatefor2-3hoursat37degCwithshakingat250rpm.
2.Transfer40mlofthecellstoasterile50mlpolypropylenecentrifugetube,andcollectthecellsbycentrifugationat3000rpmfor8minutesat4degCinaGPRcentrifuge(Beckman)or6000rpmfor8minutesat4degCinanRC5-Bcentrifuge(DuPont)equippedwithanSS-34rotor.ForM13-basedtransformation,savetheremaining10mlofcultureinanice-waterbathforlateruse.
3.Aftercentrifugation,decantthesupernatantandresuspendthecellpelletinone-halfvolume(20ml)ofcold,sterile50mMcalciumchloride,incubateinanice-waterbathfor20minutes,andcentrifugeasbefore.
4.Decantthesupernatantandgentlyresuspendthecellpelletinone-tenthvolume(4ml)ofcold,sterile50mMcalciumchloridetoyieldthefinalcompetentcellsuspension.
Preparationofcalciumchloridecompetentcellsforfrozenstorage
1.Transfer166ulofthecompetentcellsuspensiontosterileFalconculturetubes.
2.Add34ulofsterile100%glyceroltothe166ulaliquotsofthefinalcompetentcellsuspensionpreparedabove,givingafinalconcentrationof17%glycerol.
3.Thecompetentcellsthenshouldbeplacedat-70degCandcanbestoredindefinately.
4.Tousecompetentcellsfortransformation,removefromfreezerandthawforafewminutesat37degC.Placeonice,addplasmidDNAandincubateforonehourasinthestandardtransformationprocedure.Thenheatshockat42degCfor2minutes,coolbriefly,add1mlof2xTYandincubatefor1hourat37degCbeforespreadingonplates.
ElectroporationProtocol
PreparationofElectro-competentCells:
1.GrowXL1-Bluecellsonatetracyclineplate(20ugtet/mlofLBagar)
2.Inoculate3mlofYENBandgrowovernightat37degreesCwithshakingat250rpmintheNewBrunswickincubatorshaker.
3.Inoculatethe3mlofovernightgrowthinto1literofYENB(7.5gramsofBactoYeastExtractand8gramsofBactoNutrientBrothbroughtto1literwithdistilledwaterandautoclaved)andgrowtoanA600of0.5(typicallyrequires3-4hoursofshakingat250rpmintheNewBrunswickincubatorshakerat37degreesC.
4.Distributethe1literofcellsintofour500mlSorval(GS-3)centrifugebottlesandcentrifugeat5000rpmat4degreesCfor10minutes.
Note:Steps5-9shouldbeperformedinthecoldroomandtypically~600mloficecoldsterilewaterand150mloficecoldsterile10%glycerolarerequiredformanipulatingthecellsfroma1litergrowth.
5.Resuspendeachpelletin100mloficecoldsteriledoubledistilledwaterandcombinetheresuspendedpelletsintotwoSorvalcentrifugebottles(i.eeachbottlethenwillcontain200mlofresuspendedpellet).
6.Centrifugeat5000rpmat4degreesCfor10minutesintheSorvalGS-3Rotor.
7.Resuspendeachofthetwopelletsin100mloficecoldsteriledoubledistilledwaterandcombinetheresuspendedpelletsintooneSorvalcentrifugebottleandcentrifugeat5000rpmat4degreesCfor10minutesintheSorvalGS-3Rotoroncemore.Note:Thepurposeofallthesecentrifugation/resuspension/centrifugationstepsistoinsurethatthecellsareessentially"salt-free"assaltcausesarchingduringtheelectroporationstep.
8.Resuspendthepelletin100mlof10%icecoldsterileglycerol,centrifugeasabove,andfinallyresuspendthepelletin2mlof10%icecoldsterileglyceroltogivesalt-free,concentratedelectrocompetentcells.
9.Aliquote40uloftheseelectrocompetentcellsintosmallsnapcaptubesandimmediatelyfreezebyplacingincursheddryiceandthenstoreat-70degreesCuntilneeded.
ElectroporationProtocolfortransformationsusingdouble-strandedplasmids
1.Thawtheelectro-competentcellsoniceforaboutoneminute.
2.Add2-3uloftheligationmixtothecells.
3.transfer40ulofthecellsintotoBTXElectroporationcuvettesPLUSandMAKESURETHATTHECELLSCOVERTHEBOTTOMOFTHECUVETTE.
4.TurnontheBioRadE.coliPulserandsetthecurrentto2.5KVbypushingthe"Lower"and"Raise"bottomssimultaneouslytwice.
5.Placethecuvetteintheholderandslideitintoposition.
6.Chargebypressingthe"Charge"bottomuntilyouhearthebeep.
7.Immediately,suspendthecellsin1mlofYENBandtransferintoaFalcontube.
8.Incubatethecellsat37degreesCfor30minutesat250rpmshaker.
9.SpinthecellsinBECKMANtable-topcentrifugefor8minutesat2500rpm
10.Resuspendthecellsin200ulfreshYENBandadd30ulof20mg/mlXGALand30ulof25mg/mlIPTG
11.Plate~130ulofthecellsonpre-warmedLB-ampplates.
Reference:RakeshC.SharmaandRobertT.Schimke,"PreparationofElectro-competentE.coliUsingSalt-freeGrowthMedium",Biotechniques20,42-44(1996).
AbriefbackgrounddiscussionoftransformationandtransfectioncanbefoundintheAppendix.
ForDNAtransformation(14,15),theentireDNAligationreactionisaddedtoanaliquotofcompetentcells,whichismixedgently,andincubatedinanice-waterbath.Thismixturethenisheat-shockedbrieflyina42degCwaterbathfor2-5minutes.Atthispointinthetransformation,themethodvariedslightlydependingonwhetherthecloningvectorisM13-basedorpUC-based.
ForM13-basedtransformation(14),analiquotofnon-competentcellsisaddedtotheheat-shockedmixture,asisthelacoperoninducerhomologue,IPTG,andtheb-galactosidasechromogenicsubstrate,x-gal.Meltedtopagarisadded,andthetransformationmixturethenispouredontothesurfaceofanagarplate.Afterthetopagarsolidified,theplatesareinvertedandincubatedovernightat37degC.
ForpUC-basedtransformation(15),analiquotofliquidmediaisaddedtotheheat-shockedmixture,whichthenisincubatedina37degCwaterbathfor15-20minutes.Afterrecovery,thecellsuspensionisconcentratedbycentrifugationandthengentlyresuspendedinasmallervolumeoffreshliquidmedia.IPTGandx-galareaddedtothecellmixture,whichisspreadontothesurfaceofanampicillin-containingagarplate.Afterthecellmixturehaddiffusedintotheagarmedium,theplatesareinvertedandincubatedovernightat37degC.
Protocol
1.Addtheentireligationreactiontoa12X75Falcontubecontaining0.2-0.3mlofcompetentcells,mixgently,andincubateinanice-waterbathfor40-60minutes.(ForretransformationofrecombinantDNA,addapproximately10-100ngofDNAdirectlytocompetentcells).
2.Heatshockthecellsbyincubationat42degCfor2-5minutes.
ForM13-basedtransformation:
3a.Addthefollowingreagentstotheheatshockedtransformationmixture:
Non-competentcells0.2mlIPTG(25mg/mlH2O)25ulx-gal(20ml/mlDMF)25ullamBDatopagar2.5ml
4a.Mixbybrieflyvortexing,andthenquicklypourontothesurfaceofapre-warmedlambdaagarplate.
5a.Allow10-20minutesfortheagartoharden,andtheninvertandincubateovernightat37degC.
ForpUC-basedtransformation:
3b.Addthefollowingreagentstotheheatshockedtransformationmixture,add1mloffresh2xTYandincubateina37degCwaterbathfor15-30minutes.
4b.Collectthecellsbycentrifugationat3000rpmfor5minutes,decantthesupernatant,andgentlyresuspendin0.2mloffresh2xTY.
5b.Add25ulIPTG(25mg/mlwater)and25ulx-gal(20mg/mlDMF),mixandpourontothesurfaceofapre-warmedLB-Ampplate.Spreadovertheagarsurfaceusingasterilebentglassrodorsterileinoculatingloop.
6b.Allow10-20minutesfortheliquidtodiffuseintotheagar,andtheninvertandincubateovernightat37degC.
ForpBR322,pAT153orothernon-lacZcontainingvectors:
3b.Add1mloffresh2xTYtothecellsandincubatefor15-30minutesat37degC.Spreadapproximately50ulonLplatescontainingantibioticusingasterileglassspreader.Incubatetheplatesovernightat37degC.
Microcentrifugetransformationsarerecommendedwhenasingleplasmidisbeingretransformedorforqualitativetransformationexperiments.Shotguncloningexperimentsshouldbetransformedusingthelargescaletransformation,sincetheobjectiveistoefficientlyobtaintransformationofhundredsofdistinctrecombinantplasmids.
1.Inoculate50mloffresh2xTYmediawith3to5mlofafreshovernightcultureofasuitablehoststrain(GM272)andincubatefor2to3hoursat37degC.
2.Transfer1mlofthecultureintoa1.5mltubeandcentrifugefor5minatroomtemperature.Use1tubeofcultureperDNAsampletobetransformed.
3.Decantsupernatant,andresuspendthecellpelletin500ul(1/2volume)ofsterile,cold50mMcalciumchloride.Gentlyvortexifnecessary.
4.Incubate5min.onice.
5.Centrifugeasbefore,decantandresuspendthecompetentcellpelletin100ul(1/10volume)ofcalciumchloride.
6.Transfereach100ulsampleofcompetentcellstochilled12x75mmFalcontubeswhichcontain3to5ulofDNAsample(about2ng/ulto20ng/ul).
7.Incubateonicefor15minutes.
8.Heatshockthesampleat42degCfor5minutes.
9.Add1mloffresh2xTYtoeachsampleandrecoverthecellsbyincubatingat37degCfor15min.
10.ForlacZcontainingvectorsadd25ulof20mg/mlIPTG(inwater)and25ulof24mg/mlX-Gal(inDMF).
11.Add2.5mlofsofttopagartoeachsample,vortexandquicklypourontothesurfaceofaTYE-AMPagarplate.Allowatleast15-30min.fortheagartosolidify.
12.Inverttheplatesandincubateovernightat37degC.

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