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1.
A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources 总被引:5,自引:0,他引:5
A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can be performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA. 相似文献
2.
A simple technique for the obtaining of purified chromatin fractions from mammalian cells in culture is described. The procedure involves the isolation of clean nuclei in 0.30 M sucrose, 1.5 mM MgCl2, 0.2 mM CaCl2, 0.01 M Tris HCl pH 7.2, followed by sonication and differential centrifugation to collect specific chromatin fractions. Heterochromatin of SV-40 and Rous sarcoma virus transformed 3T3 cells showed a 6- to 7-fold enrichment in satellite DNA while a 2- to 3-fold enrichment in repetitive DNA was obtained in established and RSV transformed cells of Microtus agrestis. This method will facilitate the search for the site of integration of oncogenic viruses in the chromatin of mammalian cells. 相似文献
3.
An extraction procedure for the simultaneous isolation of RNA and DNA from tissue culture cells is described. The procedure is a variation of the guanidium/lithium chloride method for RNA isolation which is rapid, simple, and avoids costly ultracentrifugation equipment. The genomic DNA yielded by this procedure is greater than 50 kb in length and may be readily cleaved by restriction endonucleases. Sufficient DNA for Southern blot analysis, and RNA for Northern blot or nuclease protection analysis, can be obtained from as few as 2 x 10(6) cells, making this method particularly suitable for the genetic screening of large numbers of individual, stably transfected cell clones. 相似文献
4.
A general method for plasmid isolation in lactobacilli 总被引:10,自引:0,他引:10
Todd R. Klaenhammer 《Current microbiology》1984,10(1):23-28
A simple procedure for rapid isolation and detection of plasmid DNA fromLactobacillus species is described. Using an alkaline-detergent lysis method, plasmid DNA was released and characterized from cells treated with either mutanolysin or lysozyme for 1 h at 0°C. Treatment of cells with either enzyme at 37°C for 1h was detrimental to plasmid isolation and charaterization in someLactobacillus species. The procedure was effective with small volumes of cells and allowed rapid characterization of plasmid DNA inLactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus helveticus, andLactobacillus bulgaricus strains. 相似文献
5.
A microassay for RNA/DNA hybrids has been designed for the study of RNA from different nuclear components of Chironomus tentans salivary gland cells. The procedure comprises a scale reduction of the conventional filter method for hybridization, using ultraviolet microphotometry for quantitation of RNA and DNA. Hybridization is performed in 0.3 μl of 2 × SSC containing 1–2 × 10-2 μg DNA, immobilized on a 0.2 mm2 ‘micro-filter’, and 0.5–5 × 10−2 μg RNA, with a specific activity of more than 106 cpm/μg. Results obtained by the microtechnique are found to agree with results obtained by a large-scale, standard procedure. The applicability of the microtechnique is demonstrated in saturation and presaturation-competition experiments. RNA from micro-isolated nucleoli hybridizes a maximum of 0.22% of Chironomus tentans DNA, which corresponds to about 100 cistrons for the 38S ribosomal precursor in the haploid genome. The hybrids show a steep thermal dissociation profile with a Tm of 79 °C, close to the value expected for hybrids with a G + C content of 42%. Presaturation of filter-bound DNA by total unlabelled nucleolar RNA prevents 80% of the subsequent hybridization by labeled nucleolar Presaturation by RNA from one of the two nucleolar organizers prevents to a similar degree the subsequent hybridization by RNA from the other nucleolar organizer. This result indicates a sequence similarity of RNA transcribed in different nucleolar organizers. Further applications of the microtechnique are presented in the accompanying paper where the hybridization properties of chromosomal and nuclear sap RNA are investigated. 相似文献
6.
Isolation and biochemical characterization of nuclei from chick embryo liver 总被引:1,自引:0,他引:1
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A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl2 followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg2+ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl2. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg2+ activated RNA polymerase, 4.3 µµmoles UMP-2-C14 incorporated into RNA/µg DNA per 10 min; and Mn2+-(NH4)2SO4 activated RNA-polymerase, 136 µµmoles UMP-2-C14 incorporated into RNA/µg DNA per 45 min. 相似文献
7.
An improved procedure is presented for the binding to filter paper and subsequent purification of DNA from plasmid-containing bacterial colonies. The procedure includes treatments with NaOH, enzymatic digestion, and organic solvent extraction of the filter-bound DNA. This method allows isolation of DNA in a reusable form from thousands of colonies in several hours. Double-labeling experiments with [3H]thymidine and [14C]proline indicated that (i) during purification the DNA:protein ratio is increased several hundredfold; (ii) little or no DNA is lost during the procedure; (iii) the resultant purified DNA is tenaciously bound to the paper. Thus, the final filter-bound DNA allows multiple sequential hybridizations of different probes to one filter. 相似文献
8.
Markku Jalkanen Hannu Larjava Hilkka Turakainen Markku Tammi 《Journal of biochemical and biophysical methods》1980,3(4):195-199
An improved procedure is described for the separation and quantitation of DNA from biological samples. Following papain digestion, DNA was separated from other cell material with CdCl2 precipitation onto a glass-fiber filter and quantitated easily by the modified diphenylamine reaction. 相似文献
9.
I E Sklobovskaia A S Saenko Iu A Semin A M Poverennyi 《Biokhimii?a (Moscow, Russia)》1977,42(6):1097-1103
A radiometric method for developing the alkaline sucrose gradient sedimentation patterns of DNA from non-labeled cells is described. The method is based on labeling of DNA in the gradient fractions by means of a labeled amino acid binding to DNA in the presence of formaldehyde. The procedure involves incubation of the fractions with a mixture of the labeled amino acid and formaldehyde, filtration of the incubation mixtures through nitrocellulose filters and radiometry of the filters. Relationship between the radioactivity on the filter and DNA concentration in the sample is linear. DNA detection sensitivity and specificity are satisfactory for the use under gradient ultracentrifugation condition. The non-labeled cells DNA sedimentation patterns developed by the method described and those of DNA from the same cells labeled with 3H-thimidine in vivo are found to be similar. 相似文献
10.
The experiments undertaken in order to verify the trioxsalen-crosslinking method suggested by Russev and Vassilev for isolation of eukaryotic replication origins are described. It was found that the preparation of viral DNA isolated by the above mentioned method from CV-1 cells lytically infected with SV40 was not enriched in sequences including SV40 replication origin. The hybridization pattern of DNA preparation isolated by the trioxsalen-crosslinking procedure from chicken erythroblastosis cells with the cloned fragments of globin gene domain was found to be identical to those of the total DNA probe. The DNA fraction enriched in replication origins was isolated from the same cells with the aid of nascent DNA strand extrusion method by Zannis-Hadjopoulos et al. The hybridization pattern of this DNA fraction with the cloned fragments of chicken alpha-globin gene domain was different from those of total DNA. Taking together, the results of our experiments demonstrate that trioxsalen-crosslinking procedure does not lead to the isolation of replication origins from the objects studied in the present investigation. 相似文献
11.
A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column 总被引:4,自引:0,他引:4
A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA. 相似文献
12.
A simple technique for the isolation of very high molecular weight genomic DNA from animal tissues and cells is described. The method involves rapid isolation of nuclei and their embedding in agarose beads followed by extraction of lipids and proteins with SDS. The protocol does not require proteolytic digestion and the whole procedure can be completed in 1 day. The isolated DNA is digestible by restriction enzymes and free of ligase inhibitors. 相似文献
13.
Simplified method for isolation of white cells from whole blood suitable for direct polymerase chain reaction 总被引:1,自引:0,他引:1
A simple method for isolating mononuclear cells from whole blood is described. The procedure utilizes phytohemagglutinin to agglutinate the erythrocytes, separating white cells from whole blood in a very brief handling time. The isolated cells are readily subjected to DNA isolation simply by boiling, and the released DNA can be directly employed for the polymerase chain reaction analysis. The efficiency of this method is similar to other conventional methods, but less costly and less time-consuming. This method is particularly useful in analyzing DNA samples from the peripheral blood cells when the simplicity and low cost of the assay are preferable. 相似文献
14.
Isolation and Properties of Deoxyribonucleic Acid from Protoplasts of Cell Suspension Cultures of Ammi visnaga and Carrot (Daucus carota) 总被引:2,自引:2,他引:0
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A procedure is described for the isolation of native DNA from protoplasts of ammi (Ammi visnaga) and carrot (Daucus carota) cells. Protoplasts were produced from 40 grams of fresh cells by enzyme hydrolysis and lysed with sodium dodecyl sulfate. The DNA was purified by treatment with pronase and ribonuclease. Final isolation was achieved by sucrose density gradient centrifugation. 相似文献
15.
A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes 总被引:1,自引:0,他引:1
Tapia-Tussell R Lappe P Ulloa M Quijano-Ramayo A Cáceres-Farfán M Larqué-Saavedra A Perez-Brito D 《Molecular biotechnology》2006,33(1):67-70
A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described.
This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second,
the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A260/A280) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous
fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The
yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting
as well as for the sequence of the D1/D2 domain of the 26S rDNA. 相似文献
16.
J. Frère 《Letters in applied microbiology》1994,18(4):227-229
Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations. 相似文献
17.
A method for the rapid isolation of mitochondrial DNA from the yeast Saccharomyces cerevisiae is described. Cells are first disrupted by vortexing with glass beads and the mitochondiral DNA is then extracted directly from the cell lysate by poly-l-lysine-kieselguhr-exchange chromatography. The method is unique from most other published procedures in that there is no requirement for the isolation of either a crude or purified mitochondrial preparation. Mitochondrial DNA isolated by this procedure is shown to yield restriction endonuclease fragment patterns identical to those obtained from DNA isolated by other previously reported procedures. 相似文献
18.
A digitonin-based procedure for the isolation of mitochondrial DNA from mammalian cells 总被引:1,自引:0,他引:1
A procedure for the isolation of closed circular mitochondrial DNA (mtDNA) from cells permeabilized with digitonin is described. Compared to the standard procedure in which cells are broken after osmotic swelling, the digitonin-based procedure is more consistent and results in higher mtDNA yields. 相似文献
19.
20.
The alkaline elution technique has been modified to be used in the isolation of DNA replication intermediates and in the study of the process of DNA replication. In this procedure pulse labeled CHO cells are layered onto a membrane filter, lysed with detergent, and the nascent DNA eluted in step-wise fashion with tetrapropylammonium hydroxide at pH 11.0, 11.3, 11.5 and 12.1. Alkaline sucrose sedimentation of the eluted DNA shows that the pH 11.0 material consists of < 9S fragments consistant with those described by Okazaki and others. DNA eluting at pH 11.3 has a molecular weight of 8–12 million daltons, DNA which elutes at pH 11.5 sediments with a molecular weight of 20–30 million daltons. Two independent lines of evidence suggest that the pH 11.3 material includes DNA sequences synthesized at replicon origins. (1) Exposure of cells to low doses of X-ray prior to pulse labeling reduces the pH 11.3 fraction by 40–50% while there is little change in the other fractions. (2) Synchronization of cells by inhibiting DNA synthesis with FdU, followed by a 2 min pulse label, yields approximately 50% of the incorporated 3H-thymidine in the pH 11.3 fraction. The pH step elution technique has the following advantages: 1. Intermediates of high specific activity can be isolated from 106 cells per filter; 2. By lysing cells on a filter, proteins, nucleases, and other cellular materials are eliminated; 3. DNA in the lysate is never handled, thus eliminating shearing; 4. Eluted DNA may be instantaneously neutralized by collecting into a buffer to protect it from alkaline degradation. 相似文献