Use of lambda phasmids for deletion mapping of non-selectable markers cloned in plasmids

Dennis W. Grogan, John E. Cronan

Research output: Contribution to journalArticlepeer-review

Abstract

A nonselectable gene carried on a poorly selectable recombinant plasmid has been physically mapped by deletion analysis. Our method involved cloning the plasmid into a coliphage λ vector and treating the recombinant phage with a chelator. Virtually all particles surviving this treatment carried large deletions within the plasmid insert. Further deletion analysis was done by inserting a selectable λ sequence into one such deletion derivative and repeating the chelator selection. Chelator selection was also used to isolate deletions constructed in vitro. The deleted phage are readily characterized by restriction mapping, and the gene in question scored after infection of a mutant host strain. These techniques have enabled us to physically assign the cyclopropane fatty acid synthase gene of Escherichia coli to 0.8 kb of a 16-kb segment after characterizing only a small number of isolates. This approach should be generally useful in the mapping of plasmids for which no convenient method exists for selecting or scoring the gene in question.

Original languageEnglish (US)
Pages (from-to)75-83
Number of pages9
JournalGene
Volume22
Issue number1
DOIs
StatePublished - Apr 1983

Keywords

  • ( )
  • 4
  • Abbreviations: CFA
  • PFU
  • R broth
  • Recombinant DNA
  • SDS
  • [ ]
  • chelator selection
  • cyclopropane fatty acid
  • cyclopropane fatty acid synthase
  • deletion
  • indicates plasmid-carrier state
  • kb
  • kilobase pairs
  • ndicates prophage genome
  • phage X vectors
  • plaque-forming units
  • restriction mapping
  • see MAT & METH., section (a)
  • sodium dodecyl sulfate

ASJC Scopus subject areas

  • Genetics

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