StandardcloningforlargeinsertsandGibsonassemblyelectroporation

  • Formerly from EpicentreClonelargeinsertsandtransformlargeplasmids-uptoatleast145kbplasmidDNA.
  • GeneratecloneswithinduciblecopynumberusingCopyControl™vectors.
  • Achievehightransformationefficiencies:>1×1010cfu/µgpUC19DNA.
  • IncreaseGibsonAssembly®colonycountswithhigh-efficiencyelectroporation
FreeSampleAvailable

Applications

  • Generationofinduciblecopy-numberclonesusingtheCopyControl™CloningSystem.

TransforMax™EPI300™E.colicellslackthetonAgeneandareengineeredforusewithEpicentre'sCopyControl™cDNA,Gene,andPCRCloningKitandotherCopyControlCloningSystems*thatdonotrequirephageT1-resistantcells.ThecellscontainaninduciblemutanttrfAgenewhosegeneproductisrequiredforinitiationofreplicationfromtheoriVoriginofreplication,suchasthatinCopyControlpCC1™vectors.OnLBchloramphenicolplatesorinLBorSOCmediasupplementedwithchloramphenicol,CopyControlclonesgrowninTransforMaxEPI300E.colireplicateatsingle-copynumberfromtheF-factorrepliconbecauseexpressionofthetrfAgeneisrepressed.AdditionofCopyControlInductionSolutioninducesthecellstoexpressthetrfAgeneproductandinducestheirreplicationathigh-copynumberfromoriV(Fig.1).ReplicationfromoriVresultsinhigheryieldsandhigherpurityofclonedDNA.

Benefits

  • Copy-numberofclonesisundertightcontrolofaninduciblepromoterlinkedtothetrfAgene.
  • Hightransformationefficiencywithclonesofallsizes.
  • lacZΔM15forblue/whitescreeningofrecombinants.
  • Restriction-minus[mcrA,Δ(mrr-hsdRMS-mcrBC)]phenotypeenablesefficientcloningofmethylatedDNA.
  • Endonuclease-minus(endA1),toensurehighyieldsofDNA.
  • Recombination-minus(recA1),forgreaterstabilityoflargeinsertclones.

Figure1.CopynumberofCopyControl™BACclonescanbeinduced10-to20-foldinTransforMax™EPI300™E.coli.TheyieldofBACDNAfromCopyControlBACclonesof110-145kbincreased>14-foldfollowingadditionofCopyControlInductionSolution.U=uninducedcells;I=inducedcells.

Genotype

F-mcrAΔ(mrr-hsdRMS-mcrBC)Φ80dlacZΔM15ΔlacX74recA1endA1araD139Δ(ara,leu)7697galUgalKλ-rpsL(StrR)nupGtrfAdhfr

TransforMaxEPI300ElectrocompetentE.coli

  • Transformationefficiencyof>1x1010cfu/µgofpUC19.

TransforMaxEPI300ChemicallyCompetentE.coli

  • Transformationefficiencyof>5x108cfu/µgofpUC19.

*Coveredbyissuedand/orpendingpatents.


ORDERINFORMATION

1,000Xconcentratedsolution.Filtersterilized.