Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis

Abstract

We describe a rapid procedure for extracting purified chromosomal DNA from Pseudomonas putida, and some minor modifications needed for use in other organisms, including Gram‐positive strains. The technique is rapid and generally produces between 1 and 5 μg of DNA from 1 ml of liquid culture. The DNA is highly purified and can be readily cut with small quantities of restriction endo‐nucleases, cloned into plasmid vectors and used as a substrate for hybridization with labelled DNA probes. Genetical analysis and cloning of genomic DNA from environmentally important organisms like Ps. putida has become increasingly important. Bacterial chromosomal DNA is usually prepared from large volumes of liquid culture, and involves time‐consuming steps necessary to purify the DNA sufficiently for use in cloning experiments (e. g. Marmur 1961). We have developed a method for preparing small quantities of genomic DNA, which involves whole cell lysis and purification by precipitation and centrifugation. Loss of DNA occurs by shearing, but the yield of DNA is sufficient, and the quality is high. The method has been used to prepare DNA from a variety of organisms and is particularly applicable where the preparation and analysis of DNA from a large number of isolates is required.

Cloning and sequence analysis of the dnaK gene region of Lactococcus lactis subsp. lactis

“…Several heat-shock genes possess vegetative promoter sequences upstream of inverted repeat sequences (Table 4) and a consensus inverted repeat sequence has been described (TTAGCACTC-N,-GAGTGCTAA; Wetzstein et al, 1992), suggesting an involvement in the regulation of the heat-shock response. The precise role of the inverted repeat has not been determined but the formation of mRNA secondary structures which may protect against mRNA degradation and/or act as an activator-binding site for a DNA-or RNA-binding protein (Wetzstein et al, 1992;Li & Wong, 1992;Schmidt et al, 1992) has been proposed. In the case of the recently described dnaJ heat-shock gene of L. lactis, the stemloop is found upstream of the promoter sequence and is thought to act as a repressor-binding site.…”

“…2) which show varying degrees of complementarity to the 16s rRNA sequence of L. lactis (3'-UCUUUCCUCCA-5'; Ludwig et al, 1985). A promoter region identical to the consensus Grampositive promoter sequence (Graves & Rabinowitz, 1986) is found upstream of orfl (nucleotides 150-178) with a -35 sequence, 5'-TTGACA-3', and -10 sequence, 5'-TATAAT-3', separated by 17 bp.…”

“…Total DNA was extracted from overnight cultures of E. coli and L. lactis by the method of Lewington et al (1987). Plasmid DNA was isolated from E. coli by the alkaline lysis method (Birnboim & Doly, 1979) and all DNA manipulations were performed as described by Maniatis et al (1982).…”

The complete cps gene cluster from Streptococcus thermophilus NCFB 2393 involved in the biosynthesis of a new exopolysaccharide

“…Total DNA was extracted from Str. thermophilus as described by Lewington et al (1987). PCRs were carried out on Hybaid Omnigene thermocyclers.…”

Carnimonas nigrificans gen. nov., sp. nov., a bacterial causative agent for black spot formation on cured meat products

“…Large-scale genomic DNA isolation was performed according to Marmur (1961). Small amounts of DNA used for PCR were extracted and purified as described by Lewington et al (1987).…”