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.     

A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges

Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.     

A modified CTAB DNA extraction procedure forMusa andIpomoea

The utilization of current nucleic acid technologies in crop improvement and phylogenetic studies require the development and application of efficient DNA extraction procedures from plants. This paper describes efficient, reliable DNA extraction procedures forIpomoea andMusa. These procedures are combinations and modifications of the techniques described by Murray and Thompson (1980) and Saghai-Maroof et al. (1984) and are applicable, without further modification, to other plant genera.     

A convenient protocol for extraction and purification of DNA from Fragaria

Summary In this paper we describe a simple and efficient DNA extraction protocol for Fragaria species, a highly recalcitrant genus due to the large amount of polyphenols and polymeric carbohydrates present in strawberry tissues. The protocol yields a high quality DNA that can be amplified by polymerase chain reaction and digested with restriction endonucleases.     

Comparison of different purification procedure for extraction of staphylococcal enterotoxin A from foods.

Different procedures commonly used for extraction, purification, and concentration of staphylococcal enterotoxins from foods were investigated with 131I- and 125I-labeled staphylococcal enterotoxin A. Loss of labeled enterotoxin A was compared with loss of total nitrogen. The results showed that in most of the common procedures, such as gel filtration, ion exchange, and heat treatment, the percentage of loss of labeled enterotoxin A was greater than the loss of total nitrogen. Chloroform extraction and acid precipitation with hydrochloric acid had nearly the same effect on the purification of both labeled enterotoxin A and total nitrogen. Ammonium sulfate precipitation proved to be practical and was successfully used for purification of enterotoxin A from sausage extract. Simultaneous use of trypsin and Pseudomonas peptidase for treatment of food extracts considerably reduced food proteins capable of interfering with serological detection of enterotoxins but did not essentailly influence the loss of enterotoxin A.     

A fast and inexpensive DNA extraction/purification protocol for brown macroalgae

Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < €0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated.     

A procedure for the extraction of chloroplast DNA from broad-leaved tree species

A method for the extraction of ctDNA from isolated chloroplast was developed. This method is simple and adapted particularly to broad-leaved trees, including sclerophyllous species with high phenolic and polysaccharide contents. This method includes two major steps: first, chloroplasts are isolated in non-aqueous solutions to avoid oxidation and phenolic problems; second, ctDNA is extracted from the chloroplasts using aqueous solutions and specific methods to provide highly purified ctDNA.     

A procedure for the isolation and purification of plasmid DNA from Rhizobium meliloti

A procedure is described for the isolation and purification of the DNA of plasmids that are indigenous to the agriculturally important nitrogen-fixing bacterium Rhizobium meliloti. The procedure involves the lysis of bacteria with an ionic detergent or a mixture of ionic and nonionic detergents, the extraction of total DNA from precipitated membrane-DNA complexes, the enrichment of supercoiled plasmid DNA by the selective alkaline denaturation of chromosomal DNA, and a further purification of plasmid DNA using cesium chloridepropidium diiodide gradients. This procedure yields pure plasmid DNA in amounts of 30 to 50 μg per liter of a culture of cell density of approximately one A550 unit. The DNA thus obtained has been found to be of sufficient purity to serve as substrate for the most commonly used restriction endonucleases.     

A rapid and efficient procedure for the purification of DNA from agarose gels

DNA fragments electrophoresed through a horizontal agarose slab gel can be recovered by inserting strips of filter paper backed by dialysis membrane into slits cut in the gel in front of the DNA bands and continuing electrophoresis until the DNA is collected in the paper. Elution of the DNA from the filter paper is then achieved by low-speed centrifugation. Recovery well above 70% is routinely obtained with this technique and the DNA recovered is biologically active and can be recleaved, ligated, labeled in vitro by nick translation and hybridized to RNA.     

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3+/-2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2+/-3.8 microg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27+/-4.7 ng humic acid/microl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.     

A rapid method for extraction and purification of DNA from dental plaque.

A rapid method based on previously described DNA extraction procedures was developed for the isolation of DNA from dental plaque samples. The isolated DNA is suitable for use in the PCR. Freeze-thawing, cell wall-degrading enzymes, and guanidine isothiocyanate were used to lyse cells and release DNA. The released DNA was adsorbed onto diatomaceous earth and purified by washing with guanidine isothiocyanate, ethanol, and acetone. The purified DNA was released from the diatomaceous earth into an aqueous buffer and analyzed by PCR with 16S rDNA primers (rDNA is DNA coding for rRNA). As judged from studies with pure cultures of a number of bacterial species, gram-negative and gram-positive organisms were lysed equally well by this procedure. The amount of PCR product was proportional to the number of cells analyzed over the range tested, 500 to 50,000 cells. On the basis of studies with plaque samples that were spiked with known quantities of the oral bacterium Treponema denticola, the DNA prepared from plaque was free of substances inhibitory to PCR. This method should have utility in molecular genetic studies of bacterial populations not only in uncultured plaque samples but also in other complex bacterial assemblages.     

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.     

A rapid,cost-effective procedure for the extraction of microbial DNA from soil

Here we describe a DNA extraction method that is based on a simple, rapid polyvinylpolypyrrolidone–calcium chloride precipitation to release microorganisms from the soil combined with lysozyme–proteinase–SDS lysis of the microbial community. The extracted DNA is of high quality and allows direct detection of specific genes by the polymerase chain reaction (PCR) as well as cloning of indigenous microbial DNA. This method facilitates the extraction of 36 500-mg soil samples simultaneously in a 2-h period by one person. The procedure is safe, inexpensive, and does not require specialized equipment or generate hazardous wastes.     

A modified CTAB DNA extraction procedure for plants belonging to the family proteaceae

This paper describes rapid and efficient DNA extraction methods for mature leaves, resting buds and seedling leaves of genera in the family Proteaceae. The procedures combine and modify previously published techniques. The DNA can be digested by restriction endonucleases and is suitable for subsequent PCR amplification.     

A simple and effective separation and purification procedure for DNA fragments using Dodecyltrimethylammonium bromide

In this work we describe a simple two step separation procedure for the separation and purification of short DNA fragments. The first step involves precipitating the DNA using the cationic surfactant dodecyltrimethylammonium bromide. Dodecyltrimethylammonium bromide, unlike cetyltrimethylammonium bromide will not precipitate DNA before complexation is complete thus providing a high purity DNA. The second step involves dissolution of the DNA-dodecyltrimethylammonium complex in 75% ethanol, followed by precipitation of the Sodium-DNA salt, by titrating in a salt solution. This method is particularly suited to purification of short fragments as it does not require high salt concentrations in the ethanol precipitation step, which can be damaging for short DNA. The ability of dodecyltrimethylammonium bromide to remove ethidium bromide from intercalation sites on the DNA is also discussed