StandardcloningforlargeinsertsandT1phageresistance
AvoidphagecontaminationwithtonAmutationforresistancetobacteriophagesT1andT5.- Clonelargeinsertsandtransformlargeplasmids-uptoatleast145kbplasmidDNA.
- GeneratecloneswithinducIBLecopynumberusingCopyControl™vectors.
- Achievehightransformationefficiencies:>1×1010cfu/µgpUC19DNA.
Applications
- Constructionofinducible-copy-numbergenomiclibrariesusingtheCopyControl™CloningSystem,withclonesthatareresistanttocontaminatingphageT1andT5.
PhageT1-ResistantTransforMax™EPI300™-T1RE.colihaveallthebenefitsofthehighlyversatileTransforMaxEPI300™E.colicompetentcellswiththeadditionofbeingresistanttobacteriophagesT1andT5(tonAgenotype).Onceintroducedintothelabenvironment,bacteriophageT1rapidlylysesE.colistrainsthatarecommonlyusedincloningapplicationsandresultsinsignificantlabdowntimeandthelossofvaluableclones.BacteriophageT1isparticularlydifficulttoeliminatefromthelabandcanlaydormantformanyyears.ThetonAgenotypeprotectsthephageT1-resistantTransforMaxEC100-T1RcellsandvaluableclonesfromattackbybacteriophageT1.
LikethestandardTransforMaxEPI300E.coli,thisstrainhasbeenspeciallyengineeredforusewithEpicentre'sCopyControl™CloningSystems.*ThecellscontainaninduciblemutanttrfAgenewhosegeneproductisrequiredforinitiationofreplicationfromtheoriVcontainedontheCopyControlpCC1™VectorsoronclonesthathavebeenretrofittedwithCopyControlcapABIlityusingtheEZ-Tn5™<oriV/KAN-2>InsertionKit.ThisprocessallowsTransforMaxEPI300-T1RE.colitomaintainCopyControlvectorsatsinglecopyuntilinductionoccursimmediatelybeforeDNApurification.
Benefits
- tonAforresistancetobacteriophagesT1andT5.
- trfAgeneundertightcontrolofaninduciblepromoterforcopy-numbercontrolofCopyControlclonesandclonesretrofittedwiththeEZ-Tn5<oriV/KAN-2>Transposon.
- Hightransformationefficiencyofbothlargeandsmallclones.
- lacZΔM15forblue/whitescreeningofrecombinants.
- ReADIlyacceptslargeDNAsforconstructionoflarge-insertgenomiclibraries.
- Restrictionminus[mcrA,Δ(mrr-hsdRMS-mcrBC)]enablesefficientcloningofmethylatedDNA.
- Endonucleaseminus(endA1)toensurehighyieldsofDNA.
- Recombinationminus(recA1)forgreaterstabilityoflargeclonedinserts.
Genotype
F-mcrAΔ(mrr-hsdRMS-mcrBC)Φ80dlacZΔM15ΔlacX74recA1endA1araD139Δ(ara,leu)7697galUgalKλ-rpsL(StrR)nupGtrfAtonA
*Coveredbyissuedand/orpendingpatents.
