PhenolextractionisacommontechniqueusedtopurifyaDNAsample(1).Typically,anequalvolumeofTE-saturatedphenolisaddedtoanaqueousDNAsampleinamicrocentrifugetube.Themixtureisvigorouslyvortexed,andthencentrifugedtoenactphaseseparation.Theupper,aqueouslayercarefullyisremovedtoanewtube,avoidingthephenolinterfaceandthenissubjectedtotwoetherextractionstoremoveresidualphenol.Anequalvolumeofwater-saturatedetherisaddedtothetube,themixtureisvortexed,andthetubeiscentrifugedtoallowphaseseparation.Theupper,etherlayerisremovedanddiscarded,includingphenoldropletsattheinterface.Afterthisextractionisrepeated,theDNAisconcentratedbyethanolprecipitation.

Protocol

1.AddanequalvolumeofTE-saturatedphenoltotheDNAsamplecontainedina1.5mlmicrocentrifugetubeandvortexfor15-30seconds.

2.Centrifugethesamplefor5minutesatroomtemperaturetoseparatethephases.

3.Removeabout90%oftheupper,aqueouslayertoacleantube,carefullyavoidingproteinsattheaqueous:phenolinterface.Atthisstagetheaqueousphasecanbeextractedasecondtimewithanequalvolumeof1:1TE-saturatedphenol:chloroform,centrifugedandremovedtoacleantubeasabovebutthisadditionalextractionusuallyisnotnecessaryifcareistakenduringthefirstphenolextraction.

4.Addanequalvolumeofwater-saturatedether,vortexbriefly,andcentrifugefor3minutesatroomtemperature.Removeanddiscardtheupper,etherlayer,takingcaretoremovephenoldropletsattheether:aqueousinterface.Repeattheetherextraction.

5.EthanolprecipitatetheDNAbyadding2.5-3volumesofethanol-acetate,asdiscussedbelow.

Typically,2.5-3volumesofanethanol/acetatesolutionisaddedtotheDNAsampleinamicrocentrifugetube,whichisplacedinanice-waterbathforatleast10minutes.Frequently,thisprecipitationisperformedbyincubationat-20Covernight(1).TorecovertheprecipitatedDNA,thetubeiscentrifuged,thesupernatantdiscarded,andtheDNApelletisrinsedwithamorediluteethanolsolution.Afterasecondcentrifugation,thesupernatantagainisdiscarded,andtheDNApelletisdriedinaSpeedy-Vac.

Protocol

1.Add2.5-3volumesof95%ethanol/0.12MsodiumacetatetotheDNAsamplecontainedina1.5mlmicrocentrifugetube,inverttomix,andincubateinanice-waterbathforatleast10minutes.ItispossIBLetoplacethesampleat-20degCovernightatthisstage.

2.Centrifugeat12,000rpminamicrocentrifuge(Fisher)for15minutesat4degC,decantthesupernatant,anddraininvertedonapapertowel.

3.Add80%ethanol(correspondingtoabouttwovolumeoftheoriginalsample),incubateatroomtemperaturefor5-10minutesandcentrifugeagainfor5minutes,anddecantanddrainthetube,asabove.

4.PlacethetubeinaSavantSpeed-VacanddrytheDNApelletforabout5-10minutes,oruntildry.

5.AlwaysdissolvedriedDNAin10mMTris-HCl,pH7.6-8.0,0.1mMEDTA(termed10:0.1TEbuffer).

6.ItisadvisabletoaliquottheDNApurifiedinlargescaleisolations(i.e.100ugormore)intoseveralsmall(0.5ml)microcentrifugetubesforfrozenstoragebecauserepeatedfreezingandthawingisnotadvisable.

Notesonprecipitationofnucleicacids

A.Generalrules

Mostnucleicacidsmaybeprecipitatedbyadditionofmonovalentcationsandtwotothreevolumesofcold95%ethanol,followedbyincubationat0to-70degC.TheDNAorRNAthenmaybepelletedbycentrifugationat10to13,000xg.for15minutesat4degC.Asubsequentwashwith70%ethanol,followedbybriefcentrifugation,removesresidualsaltandmoisture.

ThegeneralprocedureforprecipitatingDNAandRNAis:

1.Addone-tenthvolumeof3MNaOAc,pH5.2*tothenucleicacidsolutiontobeprecipitated,

2.Addtwovolumesofcold95%ethanol,

3.Placeat-70degCforatleast30minutes,orat-20degCovernight.

oralternatively

1.Combine95mlof100%ethanolwith4mlof3MNaOAc(pH4.8)and1mlofsterilewater.Mixbyinversionandstoreat-20degC.

2.Add2.5volumesofcoldethanol/acetatesolutiontothenucleicacidsolutiontobeprecipitated.

3.Placeatat-70degCforatleast30minutesor-20degCfortwohourstoovernight.

*5MNH4OAc,pH7.4,NaClandLiClmaybeusedasalternativestoNaOAc.DNAalsomaybeprecipitatedbyadditionof0.6volumesofisopropanol.

B.Oligonucleotides

Addone-tenthvolumeof3MNaOAc,pH6.5,andthreevolumesofcold95%ethanol.

Placeat-70degCforatleastonehour.

C.RNA

Addone-tenthvolumeof1MNaOAc,pH4.5,and2.5volumesofcold95%ethanol.

Precipitatelargevolumesat-20degCovernight.

Smallvolumesamplesmaybeprecipitatedbyplacinginpowdereddryiceordryice-ethanolbathforfiveto10minutes.

D.IsobutanolconcentrationofDNA

DNAsamplesmaybeconcentratedbyextractionwithisobutanol.Addslightlymorethanonevolumeofisobutanol,vortexvigorouslyandcentrifugetoseparatethephases.Discardtheisobutanol(upper)phase,andextractoncewithwater-saturateddiethylethertoremoveresidualisobutanol.Thenucleicacidthenmaybeethanolprecipitatedasdescribedabove.

E.Notesonphenolextractionofnucleicacids

Thestandardandpreferredwaytoremoveproteinsfromnucleicacidsolutionsisbyextractionwithneutralizedphenolorphenol/chloroform.Generally,samplesareextractedbyadditionofone-halfvolumeofneutralized(withTEbuffer,pH7.5)phenoltothesample,followedbyvigorousmixingforafewsecondstoformanemulsion.Followingcentrifugationforafewminutes,theaqueous(top)phasecontainingthenucleicacidisrecoveredandtransferredtoacleantube.Residualphenolthenisremovedbyextractionwithanequalvolumeofwater-saturateddiethylether.Followingcentrifugationtoseparatethephases,theether(upper)phaseisdiscardedandthenucleicacidisethanolprecipitatedasdescribedabove.

A1:1mixtureofphenolandchloroformalsoisusefulfortheremovalofproteinfromnucleicacidsamples.Followingextractionwithphenol/chloroform,thesampleshouldbeextractedoncewithanequalvolumeofchloroform,andethanolprecipitatedasdescribedabove.

Restrictionenzymedigestionsareperformedbyincubatingdouble-strandedDNAmoleculeswithanappropriateamountofrestrictionenzyme,initsrespectivebufferasrecommendedbythesupplier,andattheoptimaltemperatureforthatspecificenzyme.Theoptimalsodiumchlorideconcentrationinthereactionvariesfordifferentenzymes,andasetofthreestandardbufferscontainingthreeconcentrationsofsodiumchloridearepreparedandusedwhennecessary.TypicaldigestionsincludedaunitofenzymepermicrogramofstartingDNA,andoneenzymeunitusually(dependingonthesupplier)isdefinedastheamountofenzymeneededtocompletelydigestonemicrogramofdouble-strandedDNAinonehourattheappropriatetemperature.Thesereactionsusuallyareincubatedfor1-3hours,toinsurecompletedigestion,attheoptimaltemperatureforenzymeactivity,typically37degC.SeetheAppendixforalistingofrestrictionsitespresentintheM13(pUC)MCSandalistingofvariousrestrictionenzymes,incubationconditionsandcutsites.

Protocol

1.Preparethereactionforrestrictiondigestionbyaddingthefollowingreagentsintheorderlistedtoamicrocentrifugetube:

sterileddH20q.s(where"q.s."meansquantitysufficient)10Xassaybufferone-tenthvolumeDNAxulrestrictionenzyme*yul(1-10unitsperugDNA)Totalvolumezul

*Ifdesired,morethanoneenzymecanbeincludedinthedigestifbothenzymesareactiveinthesamebufferandthesameincubationtemperature.

Note:ThevolumeofthereactiondependsontheamountandsizeoftheDNAbeingdigested.LargerDNAsshouldbedigestedinlargertotalvolumes(between50-100ul),asshouldgreateramountsofDNA.

Refertothevendor"scatalogforthechartofenzymeactivityinarangeofsaltconcentrationstochoosetheappropriateassaybuffer(10XHigh,10XMedium,or10XLowSaltBuffers,or10XSmaIBufferforSmaIdigestions).RestrictionenzymesarepurchasedfromBethesdaResearchLaboratories,NewEnglandBiolabs,orUnitedStatesBiochemicals.

2.Gentlymixbypipettingandincubatethereactionattheappropriatetemperature(typically37degC)for1-3hours.

3.Inactivatetheenzyme(s)byheatingat70-100degCfor10minutesorbyphenolextraction(seethevendor"scatalogtodeterminethedegreeofheatinactivationforagivenenzyme).Priortouseinfurtherprotocolssuchasdephosphorylationorligation,analiquotofthedigestionshouldbeassayedbyagarosegelelectrophoresisversusnon-digestedDNAandasizeMarker,ifnecessary.

Agarosegelelectrophoresis(2)isemployedtochecktheprogressionofarestrictionenzymedigestion,toquicklydeterminetheyieldandpurityofaDNAisolationorPCRreaction,andtosizefractionateDNAmolecules,whichthencouldbeelutedfromthegel.Priortogelcasting,driedagaroseisdissolvedinbufferbyheatingandthewarmgelsolutionthenispouredintoamold(madebywrappingcleartapearoundandextendingabovetheedgesofan18cmX18cmglassplate),whichisfittedwithawell-formingcomb.Thepercentageofagaroseinthegelvaried.Although0.7%agarosegelstypicallyareused,incaseswheretheaccuratesizefractionationofDNAmoleculessmallerthan1kbisrequired,a1,1.5,or2%agarosegelisprepared,dependingontheexpectedsize(s)ofthefragment(s).EthidiumbromideisincludedinthegelmatrixtoenablefluorescentvisualizationoftheDNAfragmentsunderUVlight.Agarosegelsaresubmergedinelectrophoresisbufferinahorizontalelectrophoresisapparatus.TheDNAsamplesaremixedwithgeltrackingdyeandloadedintothesamplewells.Electrophoresisusuallyisat150-200mAfor0.5-1houratroomtemperature,dependingonthedesiredseparation.Whenlow-meltingagaroseisusedforpreparativeagarosegels,electrophoresisisat100-120mAfor0.5-1hour,againdependingonthedesiredseparation,andafanispositionedsuchthattheheatgeneratedisrapidlydissipated.Sizemarkersareco-electrophoresedwithDNAsamples,whenappropriateforfragmentsizedetermination.Twosizemarkersareused,phi-X174cleavedwithrestrictionendonucleaseHaeIIItoidentifyfragmentsbetween0.3-2kbandlamBDaphagecleavedwithrestrictionendonucleaseHindIIItoidentifyfragmentsbetween2-23kb.Afterelectrophoresis,thegelisplacedonaUVlightboxandapictureofthefluorescentethidiumbromide-stainedDNAseparationpatternistakenwithaPolaroidcamera.

Protocol

1.Prepareanagarosegel,accordingtorecipeslistedbelow,bycombiningtheagarose(lowgeltemperatureagarosemayalsobeused)andwaterina500mlEhrlenmeyerflask,andheatinginamicrowavefor2-4minutesuntiltheagaroseisdissolved.

0.7%1.0%2.0%agarose1.05g1.5g3.0g20XTAE7.5ml7.5ml7.5mlddH2O142.5ml142.5ml142.5mlEtBr(5mg/ml)25ul25ul25ultotalvol150ml150ml150ml

Genetictechnologygrade(800669)orlowgeltemperature(800259)agarosefromSchwarz/MannBiotech.

2.Add20XTAEandethidiumbromide(EtBr),swirltomix,andpourthegelontoatapedplatewithcastingcombsinplace.Allow20-30minutesforsolidification.

3.Carefullyremovethetapeandthegelcastingcombsandplacethegelinahorizontalelectrophoresisapparatus.Add1XTAEelectrophoresisbuffertothereservoirsuntilthebufferjustcoverstheagarosegel.

4.Addatleastone-tenthvolumeof10XagarosegelloADIngdyetoeachDNAsample,mix,andloadintothewells.Electrophoresethegelat150-200mAuntiltherequiredseparationhasbeenachieved,usually0.5-1hour(100-120mAforlowgeltemperatureagarose),andcoolthegelduringelectrophoresiswithafan.VisualizetheDNAfragmentsonalongwaveUVlightboxandphotographwithaPolaroidcamera.

DNAfragmentsareelutedfromlow-meltingtemperatureagarosegelsusinganunpublishedprocedurefirstdevelopedbyDr.Roe.Here,thebandofinterestisexcisedwithasterilerazorblade,placedinamicrocentrifugetube,frozenat-70degC,andthenmelted.Then,TE-saturatedphenolisaddedtothemeltedgelslice,andthemixtureagainisfrozenandthenthawed.Afterthissecondthawing,thetubeiscentrifugedandtheaqueouslayerremovedtoanewtube.Residualphenolisremovedwithtwoetherextractions,andtheDNAisconcentratedbyethanolprecipitation.

Protocol

1.PlaceexcisedDNA-containingagarosegelsliceina1.5mlmicrocentrifugetubeandfreezeat-70degCforatleast15minutes,oruntilfrozen.Itispossibletopauseatthisstageintheelutionprocedureandleavethegelslicefrozenat-70degC.

2.Melttheslicebyincubatingthetubeat65degC.

3.Addone-volumeofTE-saturatedphenol,vortexfor30seconds,andfreezethesampleat-70degCfor15minutes.

4.Thawthesample,andcentrifugeinamicrocentrifugeat12,000rpmfor5minutesatroomtemperaturetoseparatethephases.Theaqueousphasethenisremovedtoacleantube,extractedtwicewithequalvolumeether,ethanolprecipitated,andtheDNApelletisrinsedanddried.

Typical5"-kinaselabelingreactionsincludedtheDNAtobelabeled,[[gamma]]-32-P-rATP,T4polynucleotidekinase,andbuffer(3).Afterincubationat37degC,reactionsareheatinactivatedbyincubationat80degC.Portionsofthereactionsaremixedwithgelloadingdyeandloadedintoawellofapolyacrylamidegelandelectrophoresed.ThegelpercentageandelectrophoresisconditionsvarieddependingonthesizesoftheDNAmoleculesofinterest.Afterelectrophoresis,thegelisdriedandexposedtox-rayfilm,asdiscussedbelowforradiolabeledDNAsequencing.

Protocol

1.Addthefollowingreagentstoa0.5mlmicrocentrifugetube,intheorderlisted:

sterileddH2Oq.s10Xkinasebuffer1ulDNAxul[[gamma]]-[32-P]-rATP10uCiT4polynucleotidekinase1ul(3U/ul)10ul

[[gamma]]-[32-P]-rATP(35020)ICNandT4polynucleotidekinase(70031)fromUnitedStatesBiochemicals.

2.Incubateat37degCfor30-60minutes.

3.Heatthereactionat65degCfor10minutestoinactivatethekinase.

FourstrainsofE.coliareusedinthesestudies:JM101forM13infectionandisolation(4),XL1BMRF"(Stratagene)forM13orpUC-basedDNAtransformation(5),andED8767forcosmidDNAtransformation(6-8).TomaintaintheirrespectiveF"episomesnecessaryforM13viralinfection(9),JM101isstreakedontoaM9minimalmedia(modifiedfromthatgiveninreference(1)plateandXL1BMRF"isstreakedontoanLBplate(1)containingtetracycline.ED8767isstreakedontoanLBplate.Theseplatesareincubatedat37degCovernight.Foreachstrain,3ml.ofappropriateliquidmediaareinoculatedwithasmearofseveralcoloniesandincubatedat37degCfor8hours,andthoseculturesthenaretransferredinto50mlofrespectiveliquidmediaandfurtherincubated12-16hours.Glycerolisaddedtoafinalconcentrationof20%,andtheglycerolstockculturesaredistributedin1.3mlaliquotsandfrozenat-70degCuntiluse(1).

Protocol

1.Streakacultureofthebacterialcellstrainontoanagarplateoftherespectivemedium,listedbelow,andincubateat37degCovernight.

E.colistrainAgarMedium/LiquidMediaXL1BMRF"(Stratagene)LB-TetJM101M9ED8767LB

2.Pickseveralcoloniesintoa12X75mmFalcontubecontaininga2mlaliquotoftherespectiveliquidmedia,andincubatefor8-10hoursat37degCwithshakingat250rpm.

3.Transferthe2mlcultureintoanEhrlenmeyerflaskcontaining50mloftherespectiveliquidmediaandfurtherincubateovernight(12-16hours)at37degCwithshakingat250rpm.

4.Add12.5mlofsterileglycerolforafinalconcentrationof20%,anddistributetheculturein1.3mlaliquotsinto12X75mmFalcontubes.

5.Storeglycerolcellstocksfrozenat-70degCuntiluse.

NotesonRestriction/ModificationBacterialStrains:

1.EcoK(alternate=EcoB)-hsdRMSgenes=attackDNAnotprotectedbyadeninemethylation.(ED8767isEcoKmethylation-).(10)

2.mcrA(modifiedcytosinerestriction),mcrBC,andmrr=methylationrequiringsystemsthatattackDNAonlywhenitISmethylated(Ed8767ismrr+,somethylatedadenineswillberestricted.Clonecancarrymethylationactivity.)(10)

3.Ingeneral,itisbesttouseastrainlackingMcrandMrrsystemswhencloninggenomicDNAfromanorganismwithmethylcytosinesuchasmammals,higherplants,andmanyprokaryotes.(11)

4.TheuseofD(mrr-hsd-mcrB)hosts=generalmethylationtoleranceandsuitABIlityforcloneswithN6methyladenineaswellas5mC(aswithbacterialDNAs).(12)

5.XL1-BlueMRF"=D(mcrA)182,D(mcrCB-hsdSMR-mrr)172,endA1,supE44,thi-1,recA,gyrA96,relA1,lac,l-,[F"proAB,lacIqZDM15,Tn10(tetr)].

HostMutationDescriptions:

araInabilitytoutilizearabinose.deoRRegulatorygenethatallowsforconstitutivesynthesisforgenesinvolvedindeoxyribosesynthesis.Allowsfortheuptakeoflargeplasmids.endADNAspecificendonucleaseI.MutationshowntoimproveyieldandqualityofDNAfromplasmidminipreps.F"F"episome,maleE.colihost.NecessaryforM13infection.galKInabilitytoutilizegalactose.galTInabilitytoutilizegalactose.gyrAMutationinDNAgyrase.Confersresistancetonalidixicacid.hflHighfrequencyoflysogeny.Mutationincreaseslambdalysogenybyinactivatingspecificprotease.lacIRepressorproteinoflacoperon.LacIqisamutantlacIthatoverproducestherepressorprotein.lacYLactoseutilization;galactosidasepermease(Mprotein).lacZb-D-galactosidase;lactoseutilization.CellswithlacZmutationsproducewhitecoloniesinthepresenceofX-gal;wildtypeproducebluecolonies.lacZdM15AspecificN-terminaldeletionwhichpermitsthea-complementationsegmentpresentonaphagemidorplasmidvectortomakefunctionallacZprotein.DlonDeletionofthelonprotease.Reducesdegradationofb-galactosidasefusionproteinstoenhanceantibodyscreeningofllibraries.malAInabilitytoutilizemaltose.proABMutantsrequireprolineforgrowthinminimalmedia.recAGenecentraltogeneralrecombinationandDNArepair.MutationeliminatesgeneralrecombinationandrendersbacteriasensitivetoUVlight.recBCDExonucleaseV.MutationinrecBorrecCreducesgeneralrecombinationtoahundredthofitsnormallevelandaffectsDNArepair.relARelaxedphenotype;permitsRNAsynthesisintheabsenceofproteinsynthesis.rspL30Sribosomalsub-unitproteinS12.Mutationmakescellsresistanttostreptomycin.AlsowrittenstrA.recJExonucleaseinvolvedinalternaterecombinationpathwaysofE.coli.strASeerspL.sbcBCExonucleaseI.PermitsgeneralrecombinationinrecBCmutants.supESupressorofamber(UAG)mutations.Somephagerequireamutationinthisgeneinordertogrow.supFSupressorofamber(UAG)mutations.Somephagerequireamutationinthisgeneinordertogrow.thi-1MutantsrequirevitaminB1(thiamine)forgrowthonminimalmedia.traD36mutationinactivatesconjugaltransferofF"episome.umuCComponentofSOSrepairpathway.uvrCComponentofUVexcisionpathway.xylAInabilitytoutilizexylose.damDNAadeninemethylase/MutationblocksmethylationofAdenineresiduesintherecognitionsequence5"-G*ATC-3"(*=methylated)dcmDNAcytosinemethylase/Mutationblocksmethylationofcytosineresiduesintherecognitionsequences5"-C*CAGG-3"or5"-C*CTGG-3"(*=methylated)hsdME.colimethylase/MutationblockssequencespecificmethylationAN6*ACNNNNNNGTGCorGCN6*ACNNNNNNGTT(*=methylated).DNAisloatedfromaHsdM-strainwillberestrictedbyaHsdR+host.hsdR17Restrictionnegativeandmodificationpositive.(rK-,mK+)AllowscloningofDNAwithoutcleavagebyendogenousrestrictionendonucleases.DNApreparedfromhostswiththismarkercanefficientlytransformrK+E.colihosts.hsdS20Restrictionnegativeandmodificationnegative.(rB-,mB-)AllowscloningofDNAwithoutcleavagebyendogenousrestrictionendonucleases.DNApreparedfromhostswiththismarkerisunmethylatedbythehsdS20modificationsystem.mcrAE.colirestrictionsystem/MutationpreventsMcrArestrictionofmethylatedDNAofsequence5"-C*CGG(*=methylated).mcrCBE.colirestrictionsystem/MutationpreventsMcrCBrestrictionofmethylatedDNAofsequence5"-G5*C,5"-G5h*C,or5"-GN4*C(*=methylated).mrrE.colirestrictionsystem/MutationpreventsMrrrestrictionofmethylatedDNAofsequence5"-G*ACor5"-C*AG(*=methylated).MutationalsopreventsMcrFrestrictionofmethylatedcytosinesequences.

OtherDescriptions:

cmrChloramphenicolresistancekanrKanamycinresistancetetrTetracyclineresistancestrrStreptomycinresistanceDIndicatesadeletionofgenesfollowingit.Tn10AtransposonthatnormallycodesfortetrTn5Atransposonthatnormallycodesforkanrspi-Referstored-gam-mutantderivativesoflambdadefinedbytheirabilitytoformplaquesonE.coliP2lysogens.CommonlyusedbacterialstrainsC600-F-,e14,mcrA,thr-1supE44,thi-1,leuB6,lacY1,tonA21,l--forplatinglambda(gt10)libraries,growswellinLbroth,2xTY,plateonNZYDT+Mg.-Huynh,Young,andDavis(1985)DNACloning,Vol.1,56-110.DH1-F-,recA1,endA1,gyrA96,thi-1,hsdR17(rk-,mk+),supE44,relA1,l--forplasmidtransformation,growswellonLbrothandplates.-Hanahan(1983)J.Mol.Biol.166,557-580.XL1Blue-MRF"-D(mcrA)182,D(mcrCB-hsdSMR-mrr)172,endA1,supE44,thi-1,recA,gyrA96,relA1,lac,l-,[F"proAB,lacIqZDM15,Tn10(tetr)]-Forplatingorglycerolstocks,growinLBwith20mg/mloftetracycline.Fortransfection,growintryptonebrothcontaining10mMMgSO4and0.2%maltose.(Noantibiotic--Mg++interfereswithtetracyclineaction.)Forpickingplaques,growglycerolstockinLBtoanO.D.of0.5at600nm(2.5hours?).Whenat0.5,addMgSO4toafinalconcentrationof10mM.SURECells-Stratagene-e14(mcrA),D(mcrCB-hsdSMR-mrr)171,sbcC,recB,recJ,umuC::Tn5(kanr),uvrC,supE44,lac,gyrA96,relA1,thi-1,endA1[F"proAB,lacIqDM15,Tn10(tetr)].Anuncharacterizedmutationenhancesthea-complementationtogiveamoreintensebluecoloronplatescontainingX-galandIPTG.GM272-F-,hsdR544(rk-,mk-),supE44,supF58,lacY1or苐acIZY6,galK2,galT22,metB1m,trpR55,l--forplasmidtransformation,growswellin2xTY,TYE,Lbrothandplates.-Hanahan(1983)J.Mol.Biol.166,557-580.HB101-F-,hsdS20(rb-,mb-),supE44,ara14,galK2,lacY1,proA2,rpsL20(strR),xyl-5,mtl-1,l-,recA13,mcrA(+),mcrB(-)-forplasmidtransformation,growswellin2xTY,TYE,Lbrothandplates.-RaleighandWilson(1986)Proc.Natl.Acad.Sci.USA83,9070-9074.JM101-supE,thi,?lac-proAB),[F",traD36,proAB,lacIqZ芃15],restriction:(rk+,mk+),mcrA+-forM13transformation,growonminimalmediumtomaintainFepisome,growswellin2xTY,plateonTYorlambdaagar.-Yanisch-Perronetal.(1985)Gene33,103-119.XL-1bluerecA1,endA1,gyrA96,thi,hsdR17(rk+,mk+),supE44,relA1,l-,lac,[F",proAB,lacIqZ芃15,Tn10(tetR)]-forM13andplasmidtransformation,growin2xTY+10礸/mlTet,plateonTYagar+10礸/mlTet(TetmaintainsFepisome).-Bullock,etal.(1987)BioTechniques5,376-379.GM2929-fromB.Bachman,YaleE.coliGeneticStockCenter(CSGC#7080);M.Marinusstrain;sexF-;(ara-14,leuB6,fhuA13,lacY1,tsx-78,supE44,[glnV44],galK2,galT22,l-,mcrA,dcm-6,hisG4,[Oc],rfbD1,rpsL136,dam-13::Tn9,xyl-5,mtl-1,recF143,thi-1,mcrB,hsdR2.)MC1000-(araD139,D[ara-leu]7679,galU,galK,D[lac]174,rpsL,thi-1).obtainedfromtheMcCarthylabattheUniversityofOklahoma.ED8767(F-,e14-[mcrA],supE44,supF58,hsdS3[rB-mB-],recA56,galK2,galT22,metB1,lac-3orlac3Y1-obtainedfromNoraHeisterkampandusedasthehostforablandbcrcosmids.

DNAfragmentslargerthanafewhundredbasepairscanbeseparatedfromsmallerfragmentsbychromatographyonasizeexclusioncolumnsuchasSephacrylS-500.Tosimplifythisprocedure,thefollowingmini-spincolumnmethodhasbeendeveloped.

1.Thoroughlymixafresh,newbottleofSephacrylS-500,distributein10mlportions,andstoreinscrewcapbottlesorcentrifugetubesinthecoldroom.

2.Priortouse,brieflyvortexthematrixandwithoutallowingtosettle,add500ulofthisslurrytoamini-spincolumn(Millipore)whichhasbeeninsertedintoa1.5mlmicrocentrifugetube.

3.Followingcentrifugationat2KRPMinatabletopcentrifuge,carefullyadd200ulof100mMTris-HCl(pH8.0)tothetopoftheSephacrylmatrixandcentrifugefor2min.at2KRPM.Repeatthissteptwicemore.PlacetheSephacrylmatrix-containingspincolumninanewmicrocentrifugetube.

4.Then,carefullyadd40ulofNEBulizedcosmid,plasmidorP1DNAwhichhasbeenendrepairedtotheSephacrylmatrix(saving2ulforlateragarosegelanalysis)andcentrifugeat2KRPMfor5minutes.Removethecolumn,savethesolutioncontainingtheeluted,largeDNAfragments(fraction1).Apply40ulof1xTMbufferandrecentrifugefor2minutesat2KRPMtoobtainfraction2andrepeatthis1xTMrinsesteptwicemoretoobtainfractions3and4.

5.TochecktheDNAfragmentsizes,load3-5ulofeacheluantfractionontoa0.7%agarosegelthatincludesascontrols,1-2ulofaPhiX174-HaeIIIdigestand2ulofunfractionated,nebulizedDNAsavedfromstep4above.

6.ThefractionscontainingthenebulizedDNAinthedesiredsizeranges(typicallyfractions1and2)areseparatelyphenolextractedandconcentratedbyethanolprecipitationpriortothekinasereaction.