Protocol for pWEBâ„¢ Cosmid Cloning Kit

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www.epicentre.com Lit.#115•8/2012 1 EPILIT115 Rev. A

pWEB™ Cosmid Cloning KitCat. No. PC8805

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pWEB™ Cosmid Cloning Kit

1. Introduction The pWEB™ Cosmid Cloning Kit provides all of the reagents needed to construct a complete and unbiased primary cosmid library in about two days using a novel cloning strategy (Fig. 1). Genomic DNA is first sheared by passing it through a standard pipette tip.ShearingtheDNAintoapproximately40-kbfragmentsleadstothehighlyrandomgeneration of DNA fragments in contrast to more biased libraries that result from fragmenting the DNA by partial restriction endonuclease digestion. The sheared DNA isend-repaired,usingreagentsinthekit,togeneratebluntendsandsizeselectedonalow-melting-pointagarosegelbycomparisonwithasupplied36-kbstandard.Finally,thesize-selectedDNAisligatedintothesuppliedblunt-endedCloning-ReadypWEBcosmidvector,packagedusingultra-highefficiencyMaxPlax™LambdaPackagingExtracts(>109 pfu/μgforphagelambda),andplatedontheincludedEPI100™-T1RPhageT1-ResistantE. coli Plating Strain.

ThebenefitsofthepWEBstrategyoverBACcloningareseveral-fold.1 First, users do not havetoisolatehighmolecularweight(HMW)DNA(>200kb),whichcanbedifficultfromsomeorganisms.HMWDNAisrequiredinthestandardapproachbecauseiftheDNAisofinsufficientlength,fewofthefragmentsgeneratedbypartialrestrictiondigestionwill have restriction sites at both ends, limiting the number of clones in the library.2 Second,inthestandardapproach,generatingoptimalsizefragmentsofgenomicDNAforcloningusingpartialrestrictiondigestsoftenrequiresextensiveoptimization.ThepWEBsystemgeneratesDNAoftheoptimalsizebyrandomshearing,thusavoidingthisdifficulty.Andthird,thedistributionofrestrictionsitesthroughoutthegenomeisrarely random; therefore following the standard approach could result in the omission of certainsequencesfromthelibrary.2 Since genomic DNA is randomly sheared in the pWEB system,itismorelikelythatallsequenceswillberepresentedinthelibrary.

pWEB (derived from pWE153)isa8179-bpcosmidcloningvectorwhichisprovidedina“ready-to-use”stateforresearcherconvenience.

ThevectorhasbeenlinearizedattheuniqueSmaIrestrictionenzymerecognitionsite,dephosphorylatedandrigorouslytestedforpurityandrecombinantcloningefficiency(Cloning-Ready).

Features of the vector include:

1) Pairs of BamH I, EcoR I, and Not I sites flank the Sma I site for excision and mapping of insert DNA.

2) ColE1originofreplicationforgrowthinE. coli.

3) Ampicillin-resistanceasanantibioticselectablemarkerforgrowthinE. coli.

4) SV40originofreplicationforgrowthineukaryoticcells.

5) Neomycin-resistanceasanantibioticselectablemarkerforgrowthineukaryoticcells.

6) Bacteriophagelambdacossiteforlambdapackagingorlambda-terminasecleavage.

7) M13ForwardPrimerbindingsiteflankingthecloningsite.

8) BacteriophageT7RNApolymerasepromoterflankingthecloningsite.

[email protected] •(800)284-8474 3


2. Product SpecificationsStorage:StoretheEPI100-T1RPhageT1-ResistantE. coliPlatingStrainandtheMaxPlaxLambdaPackagingExtractsat–70°C.Exposuretohighertemperatureswillgreatlycompromisepackagingextractefficiency.Storetheremainderofthekitcomponentsat–20°C.

Size:Reagentsincludedinthekitaresufficienttoconstruct10cosmidlibraries.

3. Kit Contents Desc. Concentration Quantity

pWEB™CosmidVector (0.5μg/μl) 20μl (Cloning-Ready;linearizedattheuniqueSma I site and dephosphorylated)End-Repair10XBuffer 100μl

(330mMTris-acetate[pH7.5],660mMpotassiumacetate,100mMmagnesiumacetate,5mMDTT)

dNTPMix (2.5mMeach) 100μlEnd-RepairEnzymeMix 50μl (including T4 DNA Polymerase and T4 Polynucleotide Kinase)

*CosmidControlDNA, (100ng/μl) 20μlATP (10mM) 100μlFast-Link™DNALigase (40U@2U/μl) 20μlFast-Link™10XLigationBuffer 100μlGELase™EnzymePreparation (25U@1U/μl) 25μlGELase™50XBuffer 200μl

(2.0MBis-Tris[pH6.0],2.0MNaCl)

AmmoniumAcetateSolution (5M) 2x1.8mlEPI100™-T1RPhageT1-ResistantE. coliPlatingStrain,glycerolstock 250μl

[F– mcrA ∆(mrr-hsdRMS-mcrBC)φ80dlacZ∆M15∆lacX74recA1endA1 araD139∆(ara, leu)7697galUgalK λ– rpsL nupG tonA]

†MaxPlax™LambdaPackagingExtracts 10ExtractsLigatedLambdaControlDNA(λc1857 Sam7) (1μ[email protected]μg/μl) 50μlControlstrainLE392MP,glycerolstock 250μl

[F– e14–(McrA–) ∆(mcrC-mrr)(TetR) hsdR514 supE44 supF58lacY1 or ∆(lacIZY)6galK2galT22metB1 trpR55 λ–]

*Note: The “T7 Control DNA” has been replaced with “Cosmid Control DNA”. The Cosmid Control DNA is used both as a size marker and as a control insert for cosmid library production. Packaged “pWEB/ Control DNA” results in colony formation as will cosmid cloning of genomic DNA.†Note: MaxPlax Lambda Packaging Extracts are supplied as freeze-thaw/sonicate extracts in unlabeled single tubes. The extracts, Ligated Lambda Control DNA, and Control Plating Strain LE392MP are packaged together in a CO2-impermeable foil pouch.

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Package &Titer

ScreenPurify GenomicDNA

Randomly Shear &End-Repair DNA

Isolate DNA of Correct SizeOptional: Concentrate Sample

Cloning-ReadypWEB

Perform Ligation

Figure 1. Production of a Cosmid Library using the pWEB™ Cosmid Cloning Kit.

Quality Control: ThepWEBCosmidCloningKitisfunction-testedusingtheprovidedCosmidControlDNA(efficienciesincolonies/μgofDNA).Eachkitmustyieldatleast107cfu/μgofCosmidControlDNA.pWEB(SmaI-Cloning-Ready)vectorpreparations,inrecircularizationassays(+ligase,–insert)mustminimallyshowa105 reduction in backgroundcolonyformationascomparedtocontrolcloning(+ligase,+insert)assaysasdescribed above.

4. Related ProductsThe following products are also available:

–pWEB-TNC™CosmidCloningKit–MasterPure™DNAPurificationKits–ColonyFast-Screen™Kits–Lambda-Terminase–EZ-Tn5™InsertionKits–T7RNAPolymerase–Plasmid-Safe™ATP-DependentDNase

5. General Considerations1. Insert DNA Quality and Quantity:Thefinalnumberofuniqueclonesinacosmid

libraryisinpartdeterminedbytheaveragesizeoftheinsertDNAmoleculesandtheconcentrationofinsertDNApresentintheligationreaction.Moststandardmethodsof isolating genomic DNA will generate a random distribution of DNA molecules, aproportionofwhichwillbeoftheoptimalsize(40kb)forligationintothepWEBvector.Occasionallyhowever,theDNAwillrequireshearingtocreatemoleculesofthis optimal length.

UsersshouldavoidexposinggenomicDNAtoUVlightandethidiumbromide.Exposureforevenshortperiodsoftime(aslittleas30seconds)candecreasetheefficiencyofcloningbytwoormoreordersofmagnitude.ThebestmethodforlocalizingDNAmoleculesinanagarosegelistostaintheDNAwithSYBR®GoldfollowedbyilluminationwithaDarkReader™Transilluminator.Usingthismethod,theDNAcanbevisualizedwhilethebandisbeingexcised,withoutlossofcloningefficiency.



2. Ligation Reaction Conditions:Thein-gelligationmethodoutlinedinpreviousversions of this protocol is no longer supported due to inconsistent performance. The new approach described in Part E of this protocol has consistently provided higher yields of clones, and is therefore strongly recommended.

3. Plating of Packaging Extracts:Theefficiencyofplatingisdependentuponseveralfactors including the growth state of host bacterial cells. We recommend a bacterial strainsuchasEPI100-T1R E. coli for infection with the packaged pWEB cosmid DNA. This bacterial strain is genetically deficient in both recombination and restriction systemstominimizetherearrangementorlossofclonesin vivo. To determine the packagingefficiencyoftheMaxPlaxLambdaPackagingExtractsonly,userscanpackage the control lambda DNA supplied in the kit and infect the control bacterial strainLE392MP.

6. Cosmid Library Construction ProtocolEPI100-T1R E. coli bacterial cells are required for the completion of this protocol.

See Part F for instructions.

A. Insert DNA Preparation

ShearingtheDNAintoapproximately40-kbfragmentsleadstothehighlyrandomgeneration of DNA fragments in contrast to more biased libraries that result from partial restrictionendonucleasedigestionoftheDNA.Frequently,genomicDNAissufficientlysheared as a result of the purification process, that additional shearing is not necessary.

1. Test the extent of shearing of the DNA by first running a small amount of it by Pulse FieldGelElectrophoresis(PFGE)(e.g.,FieldInversionGelElectrophoresis[FIGE],CHEF, etc.) with voltage and ramp times recommended by the manufacturer for separationof10-100kbDNA.IfaPFGEapparatusisnotavailable,runthesampleona20-cmlong,1%agarosegelat30-35Vovernight.Load100ngofthe36-kbCosmidControl DNA in an adjacent gel lane. Run the gel and stain with ethidium bromide.

2. If10%ormoreofthegenomicDNAmigrateswiththeCosmidControlDNA,thenproceedtoPartB.IfthegenomicDNAmigratesslower(higherMW)thantheCosmid Control DNA, then the DNA needs to be sheared further as described in step 3below.IfthegenomicDNAmigratesfasterthantheCosmidControlDNA(lowerMW)thenithasbeenshearedtoomuchandshouldbereisolated.

3. Werecommendthatatleast2.5μgandupto20μgofDNAbeusedintheshearingprocess.RandomlysheartheDNAbypassingitthrougha200-μlsmallborepipettetip.AspirateandexpeltheDNAfromthepipettetip50-100times.Examine1-2μl oftheDNAona20-cmagarosegelusingtheCosmidControlDNA.If10%ormoreof the genomic DNA migrates with the Cosmid Control DNA, then proceed to Part B. If the DNA is still too large, aspirate and expel the DNA from the pipette tip an additional 50times.Examine1-2μlofthisDNAbyagarosegelasdescribedpreviously.

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B. Insert DNA End-Repair Reaction

1. TreattheshearedDNAwiththeEnd-RepairEnzymeMixtogeneratebluntendsforcloningintothepreparedpWEBvector.Thevolumeslistedbelowareforoneend-repairreactioncontainingatotalvolumeof80μl.

2. Thawandthoroughlymixallofthereagentslistedbelowbeforedispensing;placeon ice.

Combine the following on ice:

8 μl 10XEnd-RepairBuffer 8 μl 2.5mMdNTPMix 8 μl 10mMATP x μl sterilewater 20 μgshearedinsertDNA 4 μl End-RepairEnzymeMix 80 μl Totalreactionvolume

3. Incubateatroomtemperaturefor45minutes.

4. Addgelloadingbufferandincubateat70°Cfor10min.toinactivatetheEnd-RepairEnzymeMix.

5. ProceedwithInsertDNASizeSelection(PartC).

C. Insert DNA Size Selection

1. Preparea1%lowmeltingpoint(LMP)agarosegelin1XTAEbuffer(40mMTris-acetate[pH8],1mMEDTA).

Note: Do not include ethidium bromide in the gel solution.

2. Load100ngoftheCosmidControlDNAasasizemarkerineachoftheoutsidelanesandloadtheend-repairedinsertDNAbetweenthemarkerlanes.

3. Resolvethesamplesviagelelectrophoresisatroomtemperatureovernightataconstantvoltageof30-35V.Alternatively,usersmayperformPulseFieldGelElectrophoresis or Field Inversion Gel Electrophoresis.

4. Following completion of electrophoresis, users should stain the gel with SYBR Gold according to the manufacturer’s instructions. Place the gel on a Dark Reader TransilluminatorandvisualizetheDNA.Excisea2-4mmsliceofgelcontaininginsertDNAmigratingbetweenthesizemarkers;transfertheslicetoacleantube.

5. IfaDarkReaderTransilluminatorisnotavailable,cutofftheouterlanesofthegelcontainingtheT7DNAmarker.StainthesizemarkerlaneswithethidiumbromideandvisualizetheDNAwithUVlight.Markthepositionofthesizemarkersusingapipet tip.

Note: Do not expose the insert DNA to UV irradiation, as even short exposures can decrease cloning efficiencies by 2-3 orders of magnitude. Reassemble the gel and excise a 2 to 4-mm wide gel slice containing insert DNA migrating between the size markers; transfer to a clean tube.

6. Storethegelsliceat4°Cto–20°Cforuptooneyear.

7. ProceedtoPartDwhenready.



D. Recovery of the Size-Fractionated DNA

Beforebeginningthisstep,preparea70°Canda45°Cwaterbathorothertemperatureregulated apparatus.

1. Weigh the tared tubes to determine the weight of the gel slice(s). Assume 1 mg of solidifiedagarosewillyield1μlofmoltenagaroseuponmelting(inStep2below).

2. WarmtheGELase50XBufferto45°C.MelttheLMPagarosebyincubatingthetubeat70°Cfor10-15minutes.Quicklytransferthetubeto45°C.

3. AddtheappropriatevolumeofwarmedGELase50XBufferto1Xfinalconcentration.Carefullyadd1U(1μl)ofGELaseEnzymePreparationtothetubeforeach100μlofmeltedagarose.Keepthemeltedagarosesolutionat45°Candgentlymixthesolution.Incubatethesolutionat45°Cforatleastonehour(overnightincubationcanalsobeperformedwithoutanyilleffectstotheDNA).

4. Transferthereactionto70°Cfor10minutestoinactivatetheGELaseenzyme.

5. Remove500μlaliquotsofthesolutionintosterile,1.5-mlmicrofugetube(s).

6. Chillthetube(s)inanicebathfor5minutes.Centrifugethetubesinamicrocentrifugeatmaximumspeed(~10,000rpm)for20minutestopelletanyinsolubleoligosaccharides.Any“pellet”willbegelatinous,andtranslucent-to-opaque.Carefullyremovetheupper90%-95%ofthesupernatant,which contains the DNA,toasterile1.5-mltube.Becarefultoavoidthegelatinouspellet.

7. PrecipitatetheDNA.

a) Add1/10volumeof3MSodiumAcetate(pH7.0)andmixgently.

b) Add2.5volumesofethanol.Capthetubeandmixbygentleinversion.

c) Allowprecipitationfor10minutesatroomtemperature.

d) CentrifugetheprecipitatedDNAfor20minutesinamicrocentrifuge,attop speed(typically10,000to16,000rpm).

e) Carefully aspirate the supernatant from the pelleted DNA.

f ) Washthepellet2Xwithcold,70%ethanol,repeatingstepsd)ande)usingcare not to disrupt the DNA pellet. If the pellet is dislodged, centrifuge the tube at maximum rpm to reattach.

g) Afterthesecond70%ethanolwashcarefullyinvertthetubeandallowthe pellettoair-dryfor5-10minutes(longerdrytimeswillmakeresuspensionof theDNAdifficult).

h) GentlyresuspendtheDNApelletinTEBuffer.

8. DeterminetheDNAconcentrationbyfluorimetry.Alternatively,estimatetheconcentrationoftheDNAbyrunninganaliquotoftheDNAonanagarosegelusingdilutionsofknownamountsoftheFosmidControlDNAasstandard.ManyofourcustomershavehadsuccessmeasuringtheconcentrationusingtheNanodrop®seriesofUVspectrophotometers.

Note: Measuring the DNA concentration by standard spectrophotometry (OD260) is not recommended because the DNA concentration will not be high enough to be measured accurately.

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E. Ligation Reaction

1. Please refer to Appendix A to determine the approximate number of pWEB cosmid clonesthatyouwillneedforyourlibrary.Asingleligationreactionwillproduce103-106clonesdependingonthequalityoftheinsertDNA.Basedonthisinformationcalculate the number of ligation reactions that you will need to perform. The ligation reactioncanbescaled-upasneeded.

2. Combinethefollowingreagentsintheorderlistedandmixthoroughlyaftereachaddition.A10:1molarratioofpWEBCosmidVectorinsertDNAisoptimal.

0.5μgpWEBVector≈0.09pmolvector 0.25μgof≈40kbinsertDNA≈0.009pmolinsertDNA

x μl sterilewater 1 μl 10XFast-LinkLigationBuffer 1 μl 10mMATP 1 μl pWEBVector(0.5μg/μl) x μl concentratedinsertDNA(0.25μgof≈40kbDNA) 1 μl Fast-LinkDNALigase 10 μl Totalreactionvolume

This reaction can be scaled up or down as necessary.

3. Incubateatroomtemperaturefor2hours.

4. Transferthereactionto70°Cfor10minutestoinactivatetheFast-LinkDNALigase.

ProceedtoStepForstoreat–20°C.

F. In Vitro Packaging (using MaxPlax Packaging Extracts)

Plating Bacteria Preparation:

1. Thedaybeforeperformingthepackagingreactions,inoculate50mlofLBbrothsupplementedwith10mMMgSO4and0.2%maltosewithasinglecolonyofEPI100-T1Rcellsandshakeovernightat37°C.

2. Thedayofthepackagingreactions,inoculate50mlofsupplementedLBbrothwith 5mloftheovernightcultureandshakeat37°CtoanOD600=0.8-1.0.Storethecellsat4°Cuntilneeded;cellsmaybestoredforupto72hours.

Packaging Reactions:

1. Thaw the appropriate number of packaging extracts at room temperature. For every twopackagingreactions,thawoneextract(50μl)andplaceonice.

2. Whenthawed,immediatelytransferhalf(25μl)ofeachpackagingextracttoasecond1.5-mltubeandplaceonice.

3. Add10μloftheligatedcosmidDNAtoatubecontaining25μlofextract.Ifperforminganoddnumberofpackagingreactions,theremaining25μlofextractcanberefrozenat–70°C.



4. Mixbypipettingseveraltimes;avoidtheintroductionofairbubbles.Returnallofthe contents to the bottom of the tube by brief centrifugation.

5. Incubatethereaction(s)at30°Cfor90minutes.

6. Attheendofthisincubation,addtheadditional25μlofthawedextracttoeachreactiontubeat30°Candincubatethereaction(s)foranadditional90minutesat30°C.

7. Add500μlofphagedilutionbuffer(10mMTris-HCl[pH8.3],100mMNaCl, 10mMMgCl2)andmixbygentlevortexing.Add25μlofchloroformandmixbygentlevortexingandstoreat4°C.

Titering Phage Extracts:

1. Todeterminethetiterofthepackagedcosmids,add10μlofthepackagedcosmidsto100μlofpreparedEPI100-T1R host cells. Performing a dilution series of the phage may be helpful in determining phage titers.

2. Adsorbat37°Cfor20minutes.

3. SpreadtheinfectedbacteriaonLB-ampicillinselectionplatesandincubateat37°Covernight.

4. Count colonies and calculate the titer as directed below.

Sample Calculation:

Iftherewere110coloniesontheplate,thenthetiter,cfu/ml,(wherecfurepresentscolony forming units) of this reaction would be:

(#ofcolonies)(dilutionfactor)(1000μl/ml) (110cfu)(1)(1000μl/ml) __________________________________ OR ___________________ =1.1x104cfu/ml (volumeofphageplated[μl]) (10μl)

5. Calculate the total number of clones contained within the cosmid library by multiplyingthetiter(cfu/ml)determinedinStep4abovebythetotalvolumeofthepackagedphage(e.g.,1.1x104cfu/mlx0.5ml=5.5x103 clones).

6. ThenumberofclonesrequiredtoreasonablyensurethatthecosmidlibrarycontainsenoughclonessothatanygivenDNAsequencewillbefoundinthelibrarywillvarywiththesizeofthegenome.Formostprokaryoticgenomes,afewhundredclones will be enough; for most eukaryotic genomes, several thousand clones are required.SeeAppendixAforaformulatoestimatethenumberofclonesrequired.

If the number of clones contained within the library is greater than the number of clones determinedtoensurethatagivensequenceiswithinthelibrary,thelibraryisreadytouse. See Appendices C and D for instructions regarding the Amplification and Storage of Cosmid Libraries.

If the number of clones obtained from the initial ligation and packaging reaction is lower than the desired number for the library, additional ligation and packaging reactions shouldbeperformedwiththeremainingsize-fractionatedDNAfromPartCofthisprotocol.

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7. Control DNA ReactionNote: The “T7 Control DNA” has been replaced with “Cosmid Control DNA”. The Cosmid Control DNA is used both as a size marker and as a control insert for cosmid library production. Packaged “pWEB/Control DNA” results in colony formation as will cosmid cloning of genomic DNA.

A. Control DNA Ligation into pWEB Vector

1. Combine the following reagents in the order listed and mix after each addition.

34.5 μl sterilewater 5 μl 10XFast-LinkLigationBuffer 2.5 μl 10mMATP 2 μl pWEBVector 5 μl CosmidControlDNA 1 μl Fast-LinkDNALigase 50 μl Totalreactionvolume

2. Incubateatroomtemperatureovernight.

3. Transferthereactionto70°Cfor10minutestoinactivatetheFast-LinkLigase.

4. Proceed with in vitro packaging in Part B.

B. In Vitro Packaging (using MaxPlax Packaging Extracts)

Package and titer the ligated insert DNA as directed in Part F.

Note: After spreading infected bacteria on LB-ampicillin plates, incubate the plates for a minimum of 24 hours at 37°C.

8. AppendicesAppendix A: Determining the Optimal Number of Clones in a Library

Usingthefollowingformula,2determinethenumberofcosmidclonesrequiredtoreasonablyensurethatanygivenDNAsequenceiscontainedwithinthelibrary.

N=ln(1-P)/ln(1-f )

Where P is the desired probability (expressed as a fraction); f is the proportion of the genome contained in a single clone; and Nistherequirednumberofcosmidclones.

Forexample,thenumberofclonesrequiredtoensurea99%probabilityofagivenDNAsequenceofE. colibeingcontainedwithinacosmidlibrarycomposedof40kbinsertsis:

N=ln(1-0.99)/ln(1-[4x104bases/4.7x106bases])=-4.61/-0.01=461clones



Appendix B: Testing the Efficiency of the MaxPlax Packaging Extracts

ThisprotocolcanbeusedtotestthepackagingefficiencyoftheMaxPlaxExtractsonly.The ligated lambda control DNA must be adsorbed to the control packaging strain LE392MP.

Prepareplatingbacteria(usingstrainLE392MP)asdirectedinPartF.

Perform the packaging reaction as directed in Part F.


1. Makeserialdilutionsofthepackagedphageinphagedilutionbuffer.Use10-5 and 10-6 dilutions for the control reactions.

10-2dilutionis10μlofpackagedphageparticlesinto990μlofphagedilutionbuffer;vortex mix.

10-4dilutionis10μlof10-2dilutioninto990μlphagedilutionbuffer;vortexmix.



2. Add100μloftheappropriateserialdilutionsto100μlofpreparedplatingbacteria (useLE392MPforthecontrolreactions)andincubatefor15minutesat37°C.

3. Melt0.7%topagaroseandcoolto45°C.(topagarose:LBbrothcontaining0.7%[w/v]agarosesupplementedwith10mMMgSO4.)

4. Add3mloftopagarose(45°C)totheadsorbedphage,swirl,andimmediatelypourontothesurfaceofaprewarmed(37°C)LBplate.

5. Incubatetheplatesovernightat37°C.

6. Counttheplaquesanddeterminethetiter(pfu/ml)andpackagingefficiencyasdirectedonthenextpage.MaxPlaxLambdaPackagingExtractsareguaranteedtomaintainapackagingefficiencyof1x109pfu/μgofcontrollambdaDNAforuptoone year when stored as directed.

Sample Calculations:

Iftherewere110plaquesona10-6dilutionplate,thenthetiter,pfu/ml,(wherepfurepresentsplaqueformingunits)ofthisreactionwouldbe:

(#ofplaques)(dilutionfactor)(1000μl/ml) (110pfu)(106)(1000μl/ml) __________________________________ OR _____________________ =1.1x109pfu/ml (volumeofphageplated[μl]) (100μl)

Thepackagingefficiency(pfu/μgDNA)ofthisreactionwouldbe:

(#ofplaques)(dilutionfactor)(totalreactionvol.) (110pfu)(106)(550μl) ________________________________________ OR __________________ =3x109pfu/μg (vol.ofdilutionplated)(amountofDNApackaged) (100μl)(0.2μg)



Appendix C: Amplification of Cosmid Libraries

Cosmidlibrariesaregenerallystableat4°Cforuptoseveralweekswhenstoredaspackagedphagesuspendedinphagedilutionbuffer.Forlongertermstorageortogenerate additional materials for screening, users may wish to amplify the cosmid library. Several methods are available.2Usersshouldnotehowever,thatamplificationof the library may result in a change in the composition of clones contained within the library.Thisresultsbecauseofthedifferentgrowthratesofbacteriacontainingdifferentcosmids.Asimplemethodforamplifyingcosmidlibrariesinliquidculture2 is described below.

1. Inoculate100μlofafreshovernightcultureofEPI100-T1R cells grown in LB broth supplementedwith10mMMgSO4and0.2%maltosewith10μlofpackagedphagecontaining104cosmidclones.Ifthelibrarycontainsmorethan104 clones, set up additional tubes. The addition of larger amounts of phage extract can inhibit adsorption by the bacteria. If the titer of the library is low, increase the amount of bacterialcultureproportionately.RecovertheinfectedbacteriafollowingStep3belowbycentrifugationandsuspendin100μlofLBbroth.

2. Incubateat37°Cfor20minutes.

3. Add0.5mlofLBbrothtotheinfectedcultureandincubateat37°Cfor45minutes.

4. Transfer0.5mloftheinfectedculturetothecenterofanLB-ampicillinplate(150mm).Spreadthecultureleavinga2-3mmwidestriparoundtheperimeteroftheplate.Incubateat37°Cuntilcolonies(0.2-0.3mmindiameter)appear(12-14hours).

5. Estimatethenumberofcolonies.Add10mlofLBbrothtotheplateandscrapethecoloniesoffoftheplate.Transferthebacteriatoasteriletube.Rinsetheplatewithan additional 5 ml of LB broth and transfer to the sterile tube. Transfer bacteria from additional plates to this same tube.

6. Vortexmixthetubeofbacteriatodisruptclumpsofbacterialcells.

7. Addsterileglyceroltoafinalconcentrationof15%andmixwell.Dispensethebacteriainto100to500-μlaliquotsandstoreat–70°C.

Appendix D: Amplification and Storage of Cosmid Libraries

Short Term Storage: After dilution of the packaging reaction and addition of chloroform,thepackagedcosmidlibrarycanbestoredat4°Cforseveraldays.Forlongerterm storage, see recommendations below.

Long Term Storage: For longer term storage we recommend storage of the packaged DNAasaprimarylibraryorstorageofthelibraryintheEPI300-T1RPhageT1-resistant E. coli plating strain using one of the methods described below.

Method A - Storage of Packaged DNA.

1. Tothepackagedcosmidlibrary,addsterileglyceroltoafinalconcentrationof20%,mixandstoreat–70°C.



Method B - Storage of Infected Cells.

1. Adsorb the packaged phage to bacterial cells.2. Basedontheexpectedtiter,resuspendthecellsinanappropriatevolumeofliquid

media.3. Transferthefinalresuspensiontoasteriletubeandaddsterileglyceroltoafinal

concentrationof20%.Mixthesolutionandstorealiquots(whichwouldeachconstitutealibraryofthedesiredcoverage)at–70°C.

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EPICENTRE pWEB™ Cosmid Cloning KitTM

Figure 2. pWEB Cosmid Vector.

5'...TCGTCTTCAAGAATTCGCGGCCGCCAGGGTTTTCCCAGTCACGAC

M13 Forward Primer

EcoR I Not I

GGATCCCGGGATCCTCTCCCTATAGTGAGTCGTATTATGCGGCCGCGAATTCTCA...3'

T7 Promoter Primer

BamH I EcoR INot IBamH I

Sma I

cos

col E1ori

pWEB™8179 bp

Amp R

Neo RSV40

ori

(460

)

(3467)(476

7)

(6164)

(7114)

Eco

R I

(1)

Not

I (8

)

Bam

H I

(36)

M13 ForwardPrimer T7 PrimerSm

a I (

42)

Eco

R I

(82)

Not

I (7

5)

Bam

H I

(44)

Figure 2. pWEB™ Cosmid Vector



Method C - Storage of Amplified Library.

1. Adsorb the packaged phage to bacterial cells.

2. Spreadanappropriatevolumeofinfectedbacteriaontoaplate(s)withtheappropriateantibioticandincubateat37°Covernight.

3. Add~2mlofliquidmedia(e.g.,LB)toaplateandresuspendallofthebacterialcells.

4. Transfer the resuspended cells and media to the next plate (if more than one overnight plate was used) and repeat resuspension process. Do this for as many plates as desired.

5. Transfer the final resuspension to a sterile tube and add sterile glycerol to a final concentrationof20%.Mixthesolutionandstorealiquots(whichwouldeachconstitutealibraryofthedesiredcoverage)at–70°C.

Restriction Enzymes that cut pWEB one to three times:Enzyme Sites Location AatII 1 8109AccI 1 2475AflIII 3 1428,3671,6283AhdI 1 7187ApaLI 2 6608,7854AseI 2 5627,7359AvaI 2 40,3248AvrII 1 3601BamHI 2 36,44BbvCI 1 1227BclI 1 3942BfrBI 2 3349,3421BglII 1 3937BmgBI 1 1683Bpu10I 2 1227,6094BsaI 1 7248BsaAI 2 3674,4405BsmBI 1 1312BspDI 1 108BspEI 1 5105BspLU11I 1 6283BssHII 1 4498BstBI 2 1312,4783BstEII 2 1158,1218BstXI 1 987Bsu36I 1 4975ClaI 1 108DrdI 2 4128,6402EcoNI 1 2449EcoRI 2 1,82EcoRV 1 271

Enzyme Sites Location HindIII 2 113,3617HpaI 2 1717,5824MfeI 2 926,5811MluI 1 1428MscI 1 4183NcoI 2 3508,4533NheI 1 313NotI 2 8,75NruI 3 2094,2797,4954NsiI 2 3351,3423PciI 1 6283PflFI 2 1323,4219PflMI 3 3144,3193,5260PpuMI 3 902,3262,5994PshAI 1 2539PsiI 3 5554,5573,5844PvuI 1 7557RsrII 1 4617SalI 1 2474SapI 3 4445,4655,6167ScaI 1 7667SexAI 3 1125,1438,3368SfiI 1 3554SgrAI 1 2233SmaI 1 42StuI 1 3600TatI 1 7665Tth111I 2 1323,4219XcmI 1 3917XmaI 1 40XmnI 3 840,1308,7786



Aci IAcl IAfe IAlu IAlw IAlwN IApaB IApo IAva IIBan IBan IIBbs IBciV IBfa IBfuA IBgl IBme1580IBmr IBsaB IBsaH IBsaJ IBsaW IBseY I

BsiE IBsiHKA IBsl IBsm IBSma IBsp1286IBspH IBspMIBsr IBsrB IBsrD IBsrF IBssK IBssS IBstAP IBstDS IBstF5 IBstN IBstUIBstY IBtg IBts ICac8I

CviJ IDde IDpn IDra IDsa IEae IEag IEar IEco47IIIEcoO109IFau IFnu4H IFsp IGdi IIHae IHae IIHae IIIHha IHinc IIHinf IHinP IHpa IIHph I

Hpy188IHpy99 IHpyCH4 IIIHpyCH4 IVHpyCH4 VMaeIIMaeIIIMboIMboIIMlyIMnlIMseIMslIMspIMspA1IMwoINae INar INci INgoMIVNla IIINla IVNsp I

Ple IPspG IPst IPvu IIRsa ISau3AISau96IScrF ISfaN ISfc ISfo ISim ISml ISph ISsp ISty ITaqITfi ITse ITsp45 ITsp4C ITsp509ITspR I

Acc65IAfl IIAge IAle IApa IAsc I

AsiS IBlp IBsiW IBsrG IBstZ17IDra III

Fse IKpn INde IPac IPaeR7I

Pme IPml IPspOMISac ISac II

SanD ISbf ISnaB ISpe ISrf I

Sse8647ISwa ITli IXbaIXhoI

Restriction Enzymes that cut pWEB four or more times:

Restriction Enzymes that do not cut pWEB:

AnelectroniccopyofthepWEB-TNCsequenceisavailablefordownloadingatourWebsite (http://www.epicentre.com/sequences)orcanberequestedviae-mail([email protected]) or by calling Technical Service.

pWEBCosmidVector:GenbankAccessionNo.AF075573.


MaxPlax™ Lambda Packaging Extracts

MaxPlax™ Lambda Packaging Extracts Protocol

(This protocol is also available as product literature #65 and is provided with Cat. Nos. MP5105, MP5110, and MP5120)

1. IntroductionMaxPlax™LambdaPackagingExtractsareaconvenient,high-efficiencysystemdesignedfor in vitrolambdapackagingreactions.MaxPlaxLambdaPackagingExtractsaresuppliedaspredispensedsingle-tubereactionsthathavebeenoptimizedforpackagingofmethylated and unmethylated DNA. The packaging extracts routinely yield packaging efficienciesof>1x109pfu/μgofControlλDNA.Theextractscanbeusedintheconstruction of representative cDNA libraries and genomic cloning of highly modified (methylated)DNAintoλ-phageorcosmidvectors.

Traditional packaging extracts are derived from two complementary lysogenic E. coli strains,BHB2690andBHB2688,asdescribedbyHohn(1979).5TheMaxPlaxextractsutilizeanewpackagingstrain,NM759*,reportedbyGunther,MurrayandGlazer(1993).6 This strain,whichreplacesstrainBHB2690inthepreparationofthesonicationextract,isarestriction-freeK12-derivedstraindeficientintheproductionofλ-phagecapsidproteinD.Whencombinedwiththecomplementaryfreeze-thawextractfromstrainBHB2688**,5 deficientintheproductionofλ-phagecapsidproteinE,anextremelyhigh-efficiencyofpackagingforλDNAisobtained.Moreover,theabilitytopackageλDNAbearingthemammalianmethylationpatternisgreatlyenhanced,asevidencedbythehighefficiencyofλ-vectorrescuefromtransgenicmouseDNA.6 The lack of restriction activity has beenshowntobecrucialforthehighefficiencyrescueoflambdashuttlevectorsfromtransgenic mouse DNA.6,7

*NM759: [W3110 recA56, ∆(mcrA) e14, ∆(mrr-hsd-mcr), (λimm434, clts, b2, red3, Dam15, Sam7)/λ]

**BHB2688: [N205 recA–, (λimm434 clts, b2, red3, Eam4, Sam7)/λ]

Store the MaxPlax Lambda Packaging Extracts at –70°C or below. Exposure to higher temperature will decrease packaging efficiencies.

2. Product SpecificationsStorage:StorethecontrolhostbacteriaandtheMaxPlaxLambdaPackagingExtractsat–70°C.Exposuretohighertemperatureswillgreatlycompromisepackagingextractefficiency.Avoidlongtermstorageofproductinthepresenceofdryice.Onceremovedfrom the foil package, avoid any exposure to dry ice. Store the remainder of the kit componentsat–20°C.Afterthawing,storetheControlDNAat4°C.

Storage Buffers:MaxPlaxLambdaPackagingExtractsaresuppliedasunlabeledsingletubesoffreeze-thaw/sonicateextracts.Controlplatingbacteriaaresuppliedasaglycerolstock.ControlligatedλDNAissuppliedin1XLigationBuffer.

Guaranteed Stability:MaxPlaxLambdaPackagingExtractsareguaranteedtomaintainapackagingefficiencyof>1.0x109pfu/μgofcontrolλDNA,whenstoredasdirectedforone year from the date of purchase.



3. Related ProductsThe following products are also available:

−pWEB-TNC™CosmidCloningKit− pWEB™ Cosmid Cloning Kit− EpiFOS™ Fosmid Library Production Kit− Lambda Terminase

4. Protocol for Packaging Lambda DNAThis protocol can be used for the positive control reaction as well as for experimental reactions. The positive control reactions must be plated on the control host bacterial strain(LE392MP)includedwiththeMaxPlaxExtracts.Theproperbacterialplatingstrainfor the experimental reactions will vary depending on the cloning vector used. See the vector manufacturer’s recommendations for the proper strain and plating media requirements.Ligationreactionsmaybeaddeddirectlytothepackagingextracts.Whendoingso,itisimportantto:a)addavolumeof10μlorlesstothepackagingreaction,andb)heatinactivatetheligase(i.e.,treatmentat65°Cfor15minutes)asactiveDNAligasewilldecreasepackagingefficiencies.

Solutions:

Phage Dilution Buffer LB Broth (1 Liter) LB Plates 10mM Tris-HCl(pH8.3) 10g Bacto-tryptone LBBrothwith1.5%(w/v)100mM NaCl 5g Bacto-yeastextract Bacto-agar 10mM MgCl2 10g NaCl LB Top Agar AdjustpHto7.0withNaOH LBBrothwith0.7%(w/v) Bacto-agar Plating Bacteria Preparation:

1. Thedaybeforeperformingthepackagingreactions,inoculate50mlofsupplemented(10mMMgSO4) LB broth with a single colony of the plating bacterial strainandshakeovernightat37°C.

2. Thedayofthepackagingreactions,inoculate50mlofsupplemented(10mMMgSO4+0.2%maltose)LBbrothwith5mloftheovernightcultureandshakeat37°Ctoan OD600=0.8-1.0.Storethecellsat4°Cuntilneeded;cellsmaybestoredforupto72hours.

Packaging Reactions:1. Thaw the appropriate number of packaging extracts at room temperature. For every

two packaging reactions, thaw one extract then place on ice.2. Whenthawed,immediatelytransferhalf(25μl)ofeachpackagingextracttoa

second1.5-mltubeandplaceonice.3. AddthesubstrateDNA(10μl[0.2μg]ofthecontrolDNA)toatubecontaining25μl

ofextract.Ifperforminganoddnumberofpackagingreactions,theremaining25μlofextractcanberefrozenat–70°C.



4. Mixbypipettingseveraltimes;avoidtheintroductionofairbubbles.Returnallofthe contents to the bottom of the tube by brief centrifugation if necessary.

5. Incubatethereaction(s)at30°Cfor90minutes.6. Attheendofthisincubation,addtheadditional25μlofthawedextracttoeach

reactiontubeat30°C(Ifperformingtwopackagingreactions,thawanothertubeofextractandadd25μltoeachtube.)andincubatethereaction(s)foranadditional90minutesat30°C.

7. Add500μlofphagedilutionbufferandmixbygentlevortexing.8. Add25μlofchloroformandmixbygentlevortexing(storeat4°C).9. Assaythepackagedphagebytiteringontheappropriatebacterialstrain(LE392MP

for the control).


1. Makeserialdilutionsofthepackagedphageinphagedilutionbuffer.Use10-5 and 10-6 dilutions for the control reactions.

10-2dilutionis10μlofpackagedphageparticlesinto990μlofphagedilutionbuffer;vortex mix.




2. Add100μloftheappropriateserialdilutionsto100μlofpreparedplatingbacteria (useLE392MPforthecontrolreactions)andincubatefor15minutesat37°C.

3. Add3.0mlofmeltedsupplemented(10mMMgSO4)LBtopagar(cooledto~48°C).Vortexgentlyandpourontopre-warmed(37°C)LBplates.Allowthetopagartosolidifyandthenincubateovernightat37°C.

4. Counttheplaquesanddeterminethetiter(pfu/ml)andpackagingefficiency(Seesample calculations).

Sample Calculations:

Iftherewere110plaquesona10-6dilutionplate,thenthetiter,pfu/ml,(wherepfurepresentsplaqueformingunits)ofthisreactionwouldbe:

(#ofplaques)(dilutionfactor)(1000μl/ml) (110pfu)(106)(1000μl/ml) ___________________________________ OR ______________________ =1.1x109pfu/ml (volumeofphageplated[μl]) (100μl)

Thepackagingefficiency(pfu/μgDNA)ofthisreactionwouldbe:

(#ofplaques)(dilutionfactor)(totalreactionvol.) (110pfu)(106)(550μl)________________________________________ OR ____________________ =3x109pfu/μg(vol.ofdilutionplated)(amountofDNApackaged) (100μl)(0.2μg)



5. References1. Fiandt,M.(1998)Epicentre Forum 5(3),1.2. Sambrook,J.et al.,(1989)in:Molecular Cloning: A Laboratory Manual (2nd ed.),

CSH Laboratory Press, New York.

3. Wahl,G.M.et al.,(1987)Proc. Natl. Acad. Sci. USA 84,2160.4. DiLella,A.G.andWoo,S.L.C.(1987)Meth. Enzymol. 152, 199.

5. Hohn,E.G.(1979)Methods Enzymol. 68,299.6. Gunther,E.G.et al.,(1993)Nucl. Acids Res. 21,3903.7. Kohler,S.W.et al.,(1990)Nucl. Acids Res. 18,3007.

EPI100, EpiFOS, EZ-Tn5, Fast-Link, Fast-Screen, GELase, MasterPure, MaxPlax, Plasmid-Safe, pWEB, and pWEB-TNC are trademarks of Epicentre, Madison, Wisconsin.

SYBR is a registered trademark of Molecular Probes, Inc., Eugene, Oregon.

Dark Reader is a trademark of Clare Chemical Research, Denver, Colorado.

DyNA Quant is a trademark of Hoefer Pharmacia Biotech, San Francisco, California.

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Protocol for pWEBâ„¢ Cosmid Cloning Kit