A simple and generally applicable procedure for releasing DNA from bacterial cells

Treatment of Staphylococcus simulans biovar staphylolyticus cells with acetone before digestion with lysozyme made the cells susceptible to lysis by sodium dodecyl sulfate. This technique was found to be useful for releasing DNA from a wide variety of gram-positive and gram-negative organisms.     

A method for extraction of high-quality and high-quantity genomic DNA generally applicable to pathogenic bacteria.

In this study, we report a modified procedure for extraction of high-quality genomic DNA that is rapid, simple, biologically nonhazardous, and generally applicable to pathogenic bacteria. Bacterial cells were pretreated with 70% ethanol prior to enzymatic digestion with lysozyme. Exposure of bacterial cells to 70% ethanol sterilized the cultures, making the process biologically safe and increased the susceptibility of the cells to lysozyme-induced lysis. Consistently high yields of genomic DNA (mean average yield, 0.5-2.5 mg/ml) were obtained from 465 isolates representing over 30 clinically important bacterial species. Genomic DNA obtained was determined to be suitable for further analysis, including bacterial fingerprinting techniques like restriction endonuclease analysis, Southern hybridization, and repetitive PCR. Availability of a generally applicable procedure for extraction of high-quality and high-quantity genomic DNA would be immensely beneficial for laboratories engaged in molecular surveillance of nosocomial and community-based outbreaks.     

A generally applicable cryopreservation method for nitrite-oxidizing bacteria

Nitrite-oxidizing bacteria are key members of the global nitrogen cycle but their study is hampered by their limited availability in culture, mostly due to laborious cultivation procedures and the lack of stable preservation methods. In this study, it was demonstrated that long-term cryopreservation of nitrite-oxidizing bacteria assigned to the genera Nitrobacter, Nitrospina, Nitrococcus, Nitrotoga and Nitrospira was possible using a simple and rapid protocol. Their survival was tested with different cryoprotecting agents, DMSO and Hatefi, and in various carbon-rich preservation media, ten-fold diluted TSB, and ten-fold diluted TSB supplemented with 1% trehalose, and 1% sucrose. Optimal preservation conditions were strain-dependent and marine strains appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide with or without ten-fold diluted trypticase soy broth as a preservation medium allowed successful preservation of all tested strains.     

Measurement of microgram quantities of protein by a generally applicable turbidimetric procedure

A modified turbidimetric method for protein determination which involves the use of trichloroacetic acid as the precipitating agent is described. Maximal turbidity develops in less than 30 min and is stable for at least 120 min. A linear relationship between turbidity at 340 nm and protein concentration is observed between 2 and 40 micrograms protein. Sodium dodecyl sulfate is added to avoid the interference by nonionic and cationic detergents and lipids and to decrease the protein-to-protein variation. The use of cetyltrimethyl ammonium bromide provides a two-step procedure to correct for the contribution of contaminating nucleic acid. Many compounds which interfere with other protein quantitation methods have no effect on this system. The interference of commonly used reagents as sucrose and urea can be easily corrected. This procedure compared favorably with the most widely used protein quantitation methods in simplicity, sensitivity, and specificity.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.     

Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction

A new lysis solution designated TZ, consisting of 2.0% Triton X-100 plus 2.5 mg sodium azide/ml in 0.1 M Tris-HCl buffer at pH 8.0, yielded higher levels of genomic DNA from Escherichia coli O157:H7 cells compared with a number of other commonly used cell lysis methods. Ethidium bromide stained DNA bands resulting from PCR amplification of target DNA from 100 CFU of E. coli O157:H7 were readily detected following electrophoresis of agarose gels. In contrast, conventional cell lysis methods failed to detect target DNA from 100 CFU after PCR amplification. The new solution was highly effective for lysing cell suspensions of Salmonella enteritidis, Pseudomonas putida, Lysteria monocytogenes and Psychrobacter immobilis.     

A simple procedure for isolating adenosine triphosphatase from mitochondria.

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 mumol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 mumol P/min per mg protein when measured as a release of inorganic phosphate.     

A generally applicable assay for the quantification of inhibitory effects on PCR

PCR amplification of target genes from environmental DNA extracts can suffer from PCR inhibition, caused by co-extracted substances. No simple assay has been available to quantify this inhibition. Therefore, a generally applicable PCR inhibition-assay was developed, which allows determination of statistically significant inhibition of PCR. This information is important when documenting quality of DNA in environmental extracts used as template for PCR.     

A simple and economic preservation method for genomic bacterial DNA from clinically significant pathogens

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.     

A simple procedure for maximum yield of high-quality plasmid DNA

We have established a simple procedure for the rapid isolation of high-quality plasmid DNA suitable for various molecular techniques and provided a step-by-step protocol. The DNA samples isolated by this procedure have been used successfully for double-stranded DNA sequencing, restriction enzyme mapping, subcloning, in vitro mutagenesis, generation of deletion clones and so on. The procedure is highly reproducible, and superior quality DNA can be obtained without the use of phenol, chloroform or other organic solvents.     

A simple assay procedure for beta-D-mannanase.

A simple assay procedure for beta-D-mannanase enzyme has been developed which employs carob D-galacto-D-mannan dyed with Remazolbrilliant Blue. Additionally, the procedure is quantitative, relatively sensitive, and highly specific for beta-D-mannanase enzyme. It can be readily used for the determination of beta-D-mannanase activity in crude enzyme preparations and column-chromatography eluates.