General method for plasmid DNA isolation from thermophilic lactic acid bacteria

• Published in: Journal of biotechnology Vol. 3; no. 5; pp. 323 - 332
• Main Authors: Somkuti, G.A., Steinberg, D.H.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1986; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; DNA; Fundamental and applied biological sciences. Psychology; general procedure; Genetics; isolation; lactic acid bacteria; Mission oriented research; plasmid DNA; plasmids; thermophilic lactic acid bacteria; thermophilic lactic acid bacteria; plasmid DNA; isolation; general procedure; Plasmid; Isolation; Thermophily; Lactic acid bacteria; Operating process
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/0168-1656(86)90013-1

A convenient general procedure was developed for the isolation of plasmids from thermophilic streptococci and lactobacilli that are of importance in fermented dairy foods. The protocol is a composite of selected elements of existing procedures with appropriate modifications and includes several steps for plasmid DNA enrichment. Chromosomal DNA was eliminated by differential centrifugation at 48 000 × g followed by alkali denaturation and selective precipitation in the presence of high salt and phenol extraction. The method also permitted the isolation of plasmids of various sizes (1.4–90 MDa) that are harbored by different strains of mesophilic lactic streptococci. Plasmid DNA preparations were essentially free from contaminating chromosomal DNA and protein, thus eliminating the need for density gradient centrifugation. The procedure could be used on a preparatory scale or microscale for screening large numbers of cultures for plasmid DNA content.