Factors affecting the transformation of Escherichia coli strain χ1776 by pBR322 plasmid DNA

Michael V. Norgard, Kirsten Keem, John J. Monahan

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Abstract

The susceptibility of E. coli strain χ1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0-6.0·107 cfu/ml, followed by washing twice in cold 0.1 M NaCl+5mM MgCl2+5 mM Tris, pH 7.6. Cells grown in the presence of d-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6·108 cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl+5 mM MgCl2+5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell+DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37°C or 42°C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell+DNA mixtures were plated on precooled (4°C) Tc agar plates. The efficiency of plating was optimum when only 5 μl of cell+DNA mixture was spread per plate, suggesting that non-viable background χ1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell+DNA mixtures markedly inhibited the transformation of χ1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli χ1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 107 transformants/μg pBR322 plasmid DNA.

Original languageEnglish (US)
Pages (from-to)279-292
Number of pages14
JournalGene
Volume3
Issue number4
DOIs
StatePublished - Jul 1978

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Keywords

  • Recombinant DNA
  • cycloserine
  • molecular cloning
  • vector DNA uptake

ASJC Scopus subject areas

  • Genetics

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