相关交流-蚂蚁淘在线

RandominsertionofR6KoriginforrescueofflankingDNAorplasmids

InsertR6KoriginrandomlyintoanyDNAsequenceinvitro
Skipprimerwalking-simplifySangersequencingoflargeDNAinserts
RescueclonesinE.colihostexpressingthepirgeneproduct
MinimizeinsertionbiaswiththehyperactiveTn5system,knownforhighestlevelofrandomness

Applications

"Rescue"ofplasmidsoranyothercircularizedDNA(e.g.,mitochondrialDNA)thatwouldnototherwisereplicateinE.colibecausetheylackarecognizableoriginofreplicationand/oraselectableMarker.
PreparationofDNAsequencingtemplatesfromtransposoninsertioncloneswithoutprimerwalkingoradditionalsubcloning.
Creationofalibraryofrandomgeneknockoutsinvitrotofacilitategeneticanalysisofplasmid-encodedgenes.

TheEZ-Tn5™<R6Kγori/KAN-2>InsertionKit*facilitatesthesequencingandgeneticanalysisofplasmidsoranyothercircularizedDNAthatwouldnototherwisereplicateinE.coli.^1,2ThekitisbasedupontheEZ-Tn5™<R6Kγori/KAN-2>TransposonwhichcarriestheE.coliR6Kγconditionaloriginofreplication(R6Kγori)andakanamycinresistancemarker.Asimple,one-step,2-hourinvitroreactionrandomlyinsertsthetransposonintothetargetDNA.ThenanaliquotofthereactionisusedtotransformanE.colihostexpressingthepirgeneproduct,whichisrequiredforreplicationfromtheR6Kγori.Insertionclonesareselectedonkanamycinplatesandcanbesequencedbidirectionallyusingtheprovidedprimersthatarehomologoustotheendsofthetransposon.ClonescanbemaintainedinEpicentre'sTransforMax™EC100D™pir⁺orTransforMax™EC100D™pir-116strains.³

Figure1.AplasmidcontainingtheEZ-Tn5™<R6Kγori/KAN-2>TransposoncanbemaintainedinTransforMax™EC100D™pir⁺cellsat~15copiespercell(Lanes1-4)orTransforMax™EC100D™pir-116cellsat~250copiespercell(Lanes5-8).ColoniesfromrandomlychosencloneswereprocessedusingtheColonyFast-Screen™Kit.A5-microliteraliquotoflysatewasloadedperlane.M,supercoiledDNAladder.

References

Jendrisak,J.etal.(2002)EpicentreForum9(1),14.
Yoon,Y.andKoob,M.(2003)EpicentreForum10(2),10.
Metcalf,W.W.etal.(1994)Gene138,1.

*Coveredbyissuedand/orpendingpatents,exclusivelylicensedorassignedtoEpicentre®(anIllumina®Company).

ORDERINFORMATION

EZ-Tn5™Transposase,EZ-Tn5™<R6Kyori/KAN-2>Transposon,EZ-Tn5™10XReactionBuffer,EZ-Tn5™10XStopSolution,ForwardandReversePrimers,ControlTargetDNA,SterileWater.

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