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

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

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