Isolation of Higher Molecular Weight DNA from Bacillus Cereus T Using Proteinase K

A method is described for the isolation of deoxyribonucleic acid (DNA) from Bacillus cereus T. The DNA is released from the cells autolytically in the presence of proteinase K which prevents degradation by nucleases. The method is simple and eliminates many of the previously required steps which cause mechanical shear. The DNA is of high molecular weight and can be efficiently trapped in agar to give DNA agar which is suitable for hybridization studies.     

A simple method for the direct extraction of plasmid DNA from yeast

Summary A rapid and simple method for the small scale isolation of shuttle plasmid DNA from Saccharomyces cerevisiae is described. It uses glass beads to break cells and reagents which are also used in bacterial mini-preps to yield plasmid DNA without chromosomal contamination in sufficient quantities to enable direct visualisation on agarose gels.     

Isolation of nuclei from rice tissue grown in suspension culture

Summary A simple method is described for the isolation of intact nucleic from rice tissue (Oryza sativa L.) grown in suspension culture. The procedure involves incubation of the tissue for 4 h with cellulase and pectinase prior to disruption of the cells. The yield of nucleic is approximately 40% (DNA basis) and the preparations are capable of synthesizing RNA in vitro. The method may be valuable to biochemically oriented research requiring plant nuclei.     

A simple method for the isolation of allelic series using male-linked translocations

Summary Using Adh null alleles a genetic sexing technique is being developed in Ceratitis capitata. In order to facilitate the isolation of a whole series of null alleles at this locus a technique utilizing male-linked translocations is described. It provides a simple efficient and general method for the isolation of any allelic series in species where little genetic information is available.     

A general method for plasmid isolation in lactobacilli

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.     

A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (<Emphasis Type="Italic">dahi</Emphasis>)

A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was ~50 μg/ml log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications. The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction (PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid, cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus.     

Purification of Plasma Membrane from Acanthamoeba castellanii1

A simple method for isolation of plasma membrane from Acanthamoeba using self-generating gradients of Percoll is described. To obtain a membrane marker, intact amoebae were radioiodinated and the distribution of the radiolabel was followed through the plasma membrane isolation procedure. The purity of isolated plasma membrane was assessed by enrichment of radiolabel, by electron microscopy, and by enzymatic assays for contaminating membranes. As judged from enrichment of radiolabel, a 37-fold purification of plasma membrane was obtained. We estimate that 80% of the total protein was from plasma membrane and 10% from membrane-associated actin.     

An Improved Method for the Isolation of Highly Polymerized Native Deoxyribonucleic Acid from Certain Protozoa*

A relatively simple phenol extraction method, with EDTA as the nuclease inhibitor, is described for the isolation of purified, highly polymerized native DNA from Trichomonas vaginalis, Trichomonas gallinae, and Tritrichomonas foetus; it is applicable also to Tetrahymena pyriformis. RNase Tl, RNase A (Worthington's R), pronase, and α-amylase digestions constitute important steps in obtaining satisfactory yields of DNA. High degree of polymerization of the isolation product was estimated by hyperchromicity at O.D.₂₆₀ after DNase treatment and by CsCl gradient analysis. The double-stranded condition of the DNA samples was estimated by the latter method and by denaturation with NaOH, and the molecular weight by sucrose gradient analysis. Purity of the samples was determined spectrophotometrically and by chemical analyses for protein and glycogen. DNA percent recovery was estimated by the diphenylamine reaction.     

Isolation and Properties of Isolated Nuclei from the Florida Red Tide Dinoflagellate Gymnodinium breve (Davis)1

A simple and rapid method is described for the isolation of nuclei from the Florida red tide dinoflagellate Gymnodinium breve. The nuclei are free of cytoplasmic contamination and are active in endogenous RNA synthesis. The ratio of DNA: RNA: acidsoluble protein: acid-insoluble protein is 1:0.39:0.13:0.63, respectively, and each nucleus contains ca. 113 picograms of DNA. Electrophoretic analysis of the acid-soluble proteins reveals the presence of two histone-like proteins with molecular weights of 12,000 and 13,000.     

A method for N-terminal de novo sequence analysis of proteins by matrix-assisted laser desorption/ionization mass spectrometry

A novel method for isolation and de novo sequencing of N-terminal peptides from proteins is described. The method presented here combines selective chemical tagging using succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-Ac-OSu) at the N^α-amino group of peptides after digestion by metalloendopeptidase (from Grifola frondosa) and selective capture procedures using p-phenylenediisothiocyanate resin, by which the N-terminal peptide can be isolated, whether or not it is N-terminally blocked. The isolated N-terminal peptide modified N-terminally with TMPP-Ac-OSu reagent produces a simple fragmentation pattern under tandem mass spectrometric analysis to significantly facilitate sequencing.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Extraction of high-molecular-weight DNA from dry root tissue of<Emphasis Type="Italic">Berberis lycium</Emphasis> suitable for RAPD

A simple protocol for DNA isolation from dry roots ofBerberis lycium is described. Four-year-old dry roots are used, and the isolated DNA is suitable for analysis by means of restriction enzyme digestion and random amplification of polymorphic DNA (RAPD). The method involves a modified CTAB procedure using 1% PVP to remove polysaccharides and purification using low-melting-temperature agarose. DNA is amplified by means of PCR using 10-mer random primers from Operon Biotechnologies, Inc. (USA), and DNA samples are digested withTaq I,Hind III andEcoR I and examined on agarose gels. The RAPD reaction is performed according to the 1990 protocol by Williams et al.     

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.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Microexplant isolation from Cactaceae

A method of explant isolation suitable for Cactaceae is described. Small pieces of tissue were removed with a syringe without causing substantial plant injury. Using this method callus cultures were obtained in several Cactaceae species.     

A SIMPLE METHOD FOR THE ISOLATION AND ENUMERATION OF SPARSE POPULATIONS OF CYANOBACTERIA IN ESTUARINE WATERS1

A simple, sensitive assay method for the isolation and enumeration of sparse populations of cyanobacteria in an estuarine system is described. The method, based on the standard membrane-filter plate count technique, differentiates between viable and nonviable cells. It was found that an estuarine water-based agar medium was the most suitable medium for isolation of cyanobacteria. Because of the restricted nature of colony development, isolation of individual species is easily accomplished.     

Modified method for selective enrichment and isolation of Bacillus thuringiensis from soil

Many colonies of Bacillus thuringiensis (Bt) were found to be lost using the isolation technique described by Martin and Travers modified by Morris (Morris method). Hence, a modified method for isolation of B. thuringiensis is described and compared with the Morris method. Screening methodology adopted by this method delivers an immensely rich and potentially more useful library of Bt colonies with ‘presumptive-positive’ morphology and 10-fold higher per cent frequency of Bt colonies as compared to the Morris method.     

Two new methods for recovery and genetic analysis of hybrids after fusion of yeast protoplasts

A method for regeneration of yeast protoplasts and fusants in a gelatin-agar mixture, followed by total recovery of the regenerated cells from the gelatin-agar mixture and isolation of the fusants, is described. A one-step method for obtaining intergeneric fusants in which the greater part of the genome is derived fromSaccharomyces cerevisiae, and in which the fusant can be sporulated directly and tetrad analysis carried out without construction of further hybrids, is also described.     

Preparative separation of the saponin lancemaside a from Codonopsis lanceolata by centrifugal partition chromatography

Introduction. Lancemaside A is a saponin that inhibits decreases in blood testosterone level and thus prevents or ameliorates symptoms associated with male climacteric disorder. Our initial attempt to preparative isolation of lancemaside A from the saponin fraction of Codonopsis lanceolata roots by a preparative HPLC did not give a clear result. Objective. To develop a simple and efficient method for the preparative isolation of lancemaside A from the hot water extract of C. lanceolata roots using centrifugal partition chromatography (CPC). Methodology. The saponin fraction obtained from the hot water extract of C. lanceolata roots was used as the sample for preparative‐scale separation of lancemasides by CPC using n‐hexane:n‐butanol:methanol:0.1% aqueous formic acid (3:4:1:6, v/v) as the two‐phase solvent system. The upper phase (organic phase) of the two‐phase solvent system was used as the mobile phase, and 0.5 g of saponin fraction was applied for separation by CPC. Each fraction that was separated by CPC was analysed by HPLC, and the fractions containing each of the separated compounds were pooled together, and then were purified by simple preparative HPLC. Results. The demonstrated separation sequence, hot water extraction, DIAION HP‐20 column chromatography, CPC and preparative HPLC, yielded lancemaside A, foetidissimoside A and astersaponin Hb in their pure forms. Conclusion. The simple and efficient method for the preparative isolation of lancemaside A along with two other saponins, foetidissimoside A and astersaponin Hb, from the saponin fraction of C. lanceolata was established using CPC.