Highefficiencycellstocreateuracil-containingDNAforsite-directedmutagenesis
- SolesourceofhighlyefficientElectrocompetentcells(1×109cfu/µg)
- ung-anddut-mutationstogenerateDNAwithsomeuracilresidues
- IdealgenotypeandefficiencyforKunkeldirectedmutagenesismethod1.
1KunkelT.A.1985.Rapidandefficientsite-specificmutagenesiswithoutphenotypicselection. Proc.Natl.Acad.Sci.52:488-492.
Lucigenisthesoleproviderofhigh-efficiencyelectrocompetentCJ236cells.ThiscelllinewascreatedbyaddingthepCJ105plasmidtoBW313cells2,andisideallysuitedtotheKunkeldirectedmutagenesismethod.ThismethodreliesonthecellsproducingplasmidDNAcontainingsomeuracilresidues.Thedut-andung-mutationspreventthecellsfromremovinguracilfromDNAcausedbymisincorporationordCTPdeamination.NotethatthesecellscontainanF’whichconfersresistancetoChloramphenicolatlowlevels.
AsElectrocompetentcells,thetransformationefficiencyis1×109cfu/µg.
Functionalgenotype:
[F’Tra+Pil+(CamR)]ung-1relA1dut-1thi-1spoT1mcrA
2Joyce,C.M.,N.Grindley1984.MethodfordeterminingwhetherageneofEscherichiacoliisessential:applicationtothepolAgene.J.Bacteriol.158:636-643
