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Nucleic acid alcohol precipitation

In a number of methods, isolation of the nucleoprotein complex (stage 2) is avoided. In the isolation of ribonucleic acid from beef pancreas,1241 nuclear material and cell debris are removed from a normal-saline extract of the minced tissue, which is then brought to half-saturation with sodium chloride (to dissociate the protein from the nucleic acid). After removal of the protein, the nucleic acid is precipitated with alcohol. However, the suggestion has been made126 that it is more satisfactory to isolate the nucleoprotein first, and this has been carried out, for instance, in the extraction of the ribonucleic acid from fowl sarcoma GRCH 15.126 Nucleoprotein complexes have also been isolated from baker s yeast127 and have been separated into various fractions, the nucleic acids from which differ slightly in composition. In addition, nucleoproteins have been isolated by complex formation with cetyltrimethylammonium bromide.128... [Pg.309]

For very dilute nucleic acid solutions, precipitation with polyethylene glycol (PEG) is preferred. PEG 6000 is added to a concentration of 10% (w/v), and the solution is allowed to stand on ice for 2 h. The precipitate is removed by centrifugation and washed with 70% (v/v) ethanol. PEG can be used for fractional precipitation, since high molecular weight DNA is precipitated at lower PEG concentrations than low molecular weight DNA. It should be emphasised that oligonucleotides of chain length less than 20 cannot be precipitated effectively with either alcohol or PEG. [Pg.61]

DNA Extraction and Analysis. Intracellular extrachromosomal HBV DNA was extracted from cells by the method of Hirt (256) as adopted by Gripon et al. (257). Briefly, cells were lysed in buffer containing 0.5% (w/v) sodium dodecyl sulfate (SDS), 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 10 mM EDTA and 200 /ig/ml proteinase K, and incubated overnight at 37°C. NaCl was added to the lysate to a final concentration of 1 M, and the mixture was stored overnight at 4 C to allow cellular DNA to precipitate. Chromosomal DNA was pelleted at 29,(XX) X g for 1 hr at 4°C. The supernatant was extracted twice with an equal volume of phenol chloroform isoamyl alcohol (25 24 1, v/v/v) followed by two extractions with chloroform. isoamyl alcohol (24 1, v/v). Sodium acetate (pH 5.5) was added to the aqueous phase to a final concentration of 0.3 M, and nucleic acids were precipitated with 2 volumes of ethanol. Pellets recovered after centrifugation at 29,000 x g for 10 min were dissolved in 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA. [Pg.537]

Nucleic acids are water soluble because of the polyanionic character of the molecule. It is possible to obtain viscous aqueous solutions of DNA up to 1 % (w/ v). The long, thin DNA structure means that the molecule is very susceptible to cleavage by shearing or sonication in solution, which results in a reduction in solution viscosity. The nucleic acids are precipitated from aqueous solution by the addition of alcohol. [Pg.198]

The upper aqueous phase containing nucleic acids is then separated and the DNA precipitated by addition of ethanol. Because of the ionic nature of DNA, it becomes insoluble if the aqueous medium is made less polar by addition of an organic solvent. The DNA forms a threadlike precipitate that can be collected by spooling onto a glass rod. The isolated DNA may still be contaminated with protein and RNA. Protein can be removed by dissolving the spooled DNA in saline medium and repeating the chloroform-isoamyl alcohol treatment until no more denatured protein collects at the interface. [Pg.404]

Highly purified DNA may be obtained by repeating the chloroform-isoamyl alcohol extraction several times. The alcohol precipitation step may also be carried out many times. Various chromatographic methods including ion exchange have been applied to the purification of nucleic acids. [Pg.464]

Once the nucleic acids are in solution, they must be separated from the other constituents of the cell. First, the protein molecules must be removed. Many of the proteins of the cell are strongly associated with nucleic acids. The addition of sodium perchlorate (NaC104) dissociates the proteins from nucleic acids. When the mixture is shaken with the organic solvent, chloroform-isoamyl alcohol, the proteins are denatured, and they precipitate at the interface. At the same time, the lipid components of the cells are dissolved in the organic solvent. Thus the aqueous layer will contain nucleic acids, small water-soluble molecules, and even some proteins as contaminants. [Pg.467]

Alcohol is by far the most important predpitation agent used with nucleic acids. Predpitation is usually carried out with 2-3 (v/v) of ethanol or 1 (v/v) of isopropanol in the presence of 0.1-0.5 M Na or K acetate at pH 5.0 and 0 °C salt concentrations higher than 1 M interfere with precipitation. Monovalent cations and ethanol produce a conformational change in the nudeic add which leads to precipitation. For quantitative predpitation, the mixture should be kept for 15 min at -70 °C or 30 min at -20 °C. This is particularly important for dilute solutions (< 10 pg ml-1) more concentrated solutions, above about 0.25 mg ml-1, are predpitated quantitatively even at room temperature. Sodium and potassium acetate salts in the mixture are partially predpitated with the nuddc acid, but they can be removed by washing... [Pg.60]

Peptic and tryptic digestion of Type I pneumococcal cells, followed by alcoholic precipitation and subsequent removal of the nucleic acid, gave SI in 92-98% purity. Hydrolysis (with N sulfuric acid at 100°) liberated galactose and fucose within 15 minutes, followed by 2-amino-2-deoxy-D-glucose (in one hour) and galacturonic acid (in 6-8 hours). Quantitative immunological data were not given for the preparation. [Pg.309]

Nucleoprotein complex can be extracted from cells with N NaCl solution. If the resulting viscous solution is shaken with chloroform containing a little cetyl alcohol, the protein forms a gel at the chloroform/water interface and the sodium salts of the nucleic acids remain in the aqueous phase. Chromatography and centrifuging can then be used to isolate pure specimens (Chapter 14.3). Samples of DNA can be dissolved in water to form very viscous solutions. On adding alcohol to these, a soggy cotton wool-type of precipitate is obtained, from which semi-crystalline threads of DNA can be picked out. [Pg.884]


See other pages where Nucleic acid alcohol precipitation is mentioned: [Pg.346]    [Pg.47]    [Pg.71]    [Pg.47]    [Pg.409]    [Pg.409]    [Pg.279]    [Pg.316]    [Pg.352]    [Pg.174]    [Pg.209]    [Pg.67]    [Pg.299]    [Pg.295]    [Pg.52]    [Pg.58]    [Pg.313]    [Pg.315]    [Pg.57]    [Pg.237]    [Pg.267]    [Pg.1400]    [Pg.1400]    [Pg.328]    [Pg.47]    [Pg.415]    [Pg.143]    [Pg.237]    [Pg.34]    [Pg.105]    [Pg.4]    [Pg.297]    [Pg.297]    [Pg.71]    [Pg.128]    [Pg.465]    [Pg.346]   
See also in sourсe #XX -- [ Pg.60 ]




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