A general method of isolating high molecular weight DNA from methanogenic archaea (archaebacteria).

High molecular weight DNA was readily isolated from all methanogens treated, as well as from thermophilic anaerobic eubacteria, by grinding cells frozen in liquid N2, prior to lysis with SDS. DNA can subsequently be purified by the usual phenol-chloroform extractions. The procedure yields DNA readily cut by restriction enzymes and suitable for oligonucleotide probing, as well as for mole percent G + C content determination by thermal denaturation. The method routinely yields DNA of high molecular weight and is an improvement over DNA isolation methods for many methanogens, which often involve an initial breakage of the cells in a French pressure cell.     

A rapid, efficient method for isolating DNA from yeast

A method is described for the purification of chromosomal and plasmid DNA from the yeast Saccharomyces cerevisiae. This method is rapid, gives 75% of theoretical yield, and produces DNA that can be cut with restriction endonucleases. Yeast cells are treated with zymolyase, and the resulting spheroplasts are lysed in the presence of the chaotropic agent guanidine hydrochloride. After a brief ethanol precipitation, protein is removed by treatment with proteinase K followed by phenol-chloroform extraction. After ethanol precipitation, the DNA is sufficiently pure for restriction analysis or for the transformation of Escherichia coli.     

A general method for isolation of high molecular weight DNA from eukaryotes.

A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.     

A simple and efficient method for isolating trichomes for downstream analyses

Arabidopsis trichomes are an excellent cell type to address many questions in plant biology including the control of cell shape, endoreplication, and cell expansion. Because trichomes comprise such a small percentage of the cells of a leaf, biochemical analyses of trichomes are limited. To overcome this limitation, we developed a method for removing trichomes from the leaf surface. Our method allows the isolation of intact trichomes for use in downstream applications such as cell wall analysis, immunolocalization of trichome proteins, analysis of DNA content, and proteomics. Also, this method will facilitate the isolation of trichomes from practically any plant species.     

A simple method for the determination of DNA molecular weight distribution

A method is described for the molecular weight distribution of DNA which is determined from sedimentation-velocity analysis. Knowing the distribution of sedimentation coefficients for a single DNA concentration it is possible to extrapolate such a distribution to infinite dilution of the solute in a simple way. Two versions (using two or three terms of a series) of extrapolating equations are considered and discussed in detail. The sedimentation coefficients distribution calculated from these equations differs only insignificantly with that obtained in a conventional way.     

Simple and efficient protocol for isolation of high molecular weight DNA from Streptomyces aureofaciens

A simple and efficient protocol involving the use of cetyltrimethylammonium bromide (CTAB) for rapid isolation of high molecular wt ( 50 kb) DNA from Streptomyces aureofaciens is described. The DNA yields range from 1.5–2.5 mg per 1.0 g (wet wt) of mycelia, with the purity measured at A260/280 of 1.83–1.97 and also at A260/230 of 2.2–2.71. The DNA preparation is suitable as substrate for restriction digestion, Southern hybridization and library construction. © Rapid Science Ltd. 1998     

A method for the preparation of high molecular weight yeast DNA

A method is described for the isolation of high molecular weight DNA in solution using the principles that have allowed electrophoresis of chromosome-sized DNA in pulse field gradient electrophoresis. Stationary phase yeast cells are converted to spheroplasts by the action of zymolyase in 1 M sorbitol. In the presence of EDTA and sodium lauroyl sarcosinate, proteins are digested with proteinase K. DNA is extracted with phenol and chloroform, and high molecular weight DNA is collected by ethanol precipitation. RNA is removed by RNase digestion of the redissolved pellet, and RNase is removed by chloroform extraction followed by a second ethanol precipitation. The method is rapid and gives a high yield of DNA that is readily digestible by restriction endonucleases.     

A simple and efficient method for chemical mutagenesis of DNA.

A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA segment is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragment is obtained. This methoxylamine-derivatized DNA segment is then joined with linearized M13 RF DNA, competent E. coli is transfected, and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM beta-lactamase.     

A simple method for isolating osteoblasts from mouse calvaria

A method for isolating osteoblasts from mouse calvaria, based on the capability of these cells to migrate onto plastic substrates, is reported. The osteoblasts migrate from small (1-2 mm2) regularly cut fragments of calvaria directly onto plastic surface as a continuous cellular sheet. Isolated osteoblasts retain a poligonal shape and the ability to initiate bone matrix formation in presence of B-glycero phosphate. The method is simple and provides a large quantity of osteoblasts ready to be used directly without the need of being detached and reseeded.     

Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin

We describe a new technique designed to isolate specific eukaryotic genes as native oligonucleosome fragments. The isolation method consists of hybridization of single-stranded termini of chromatin restriction fragments to complementary mercurated DNA probes, followed by isolation of the hybrids by sulfhydryl-Sepharose chromatography. SV40 minichromosomes were used to test the effectiveness of the technique. About 80% of KpnI- or BamHI-restricted and lambda exonuclease treated SV40 minichromosomes hybridized to an appropriate DNA probe after a 12-h hybridization reaction under mild conditions (0.1 M aqueous salt, 37 degrees C, pH 8). When the restricted minichromosomes were mixed with a 15-fold excess of "background" chromatin from sea urchin embryos, nucleoprotein hybridization was able to reisolate the SV40 chromatin to 88% purity with a 63% yield. This represented a 115-fold enrichment of specific genes as chromatin. Results of electron microscopy and polyacrylamide gel electrophoresis indicate that the hybridized SV40 chromatin has not lost the major chromosomal proteins characteristic of SV40 nor acquired significant amounts of protein due to exchange with background chromatin. Our experimental results show that it is currently possible to isolate repeated genes from higher eukaryotes for structural and biochemical study of the proteins involved with gene regulation.     

Glycogen of high molecular weight from mammalian muscle

Glycogen of high molecular weight has been isolated from mammalian muscle, in contrast to the material of low molecular weight commonly described. The large polysaccharide is similar to liver glycogen in the structure of its individual beta-particles and also, partially, in the mode of assembly into the gross alpha-particles. The large particles may be disrupted by 2-mercaptoethanol, but not to the same extent as their liver counterparts.     

A simple and efficient method for isolation of DNA in high mucilaginous plant tissues

A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was used with healthy Bixa orellana and virus-infected Malvaceae plants.