Deproteinization of bacterial genomic and plasmid DNAs by tannic acid.

Tannic acid is proposed as a protein precipitating agent in genomic and plasmid DNAs preparation. After addition of tannic acid, a single centrifugation step is sufficient to deproteinize Escherichia coli and Serratia marcescens extracts. The purified DNA lent itself to amplification, restriction enzymes digestion and transformation. Tannic acid is much less toxic than common deproteinizing agents and it is environmentally friendly, biodegradable, renewable and quite inexpensive.

Effects of addition of straw, chitin and manure to new or recycled biofilters on their pesticides retention and degradation properties.

Pollution of surface and groundwater by pesticides is an increasing problem that needs to be addressed by the authorities as well as by the farmers themselves. Nowadays, some researchers are considering the numerous small spillages at the farm sites as a relevant entry route to be taken into account for predicting surface and groundwater pollution. In order to tackle this problem, several solutions exist for limiting the disposal of pesticide wastes into the environment. One such system is biopurification of farm wastes by biobed, biofilter or phytobac. In this study, the results of pesticides retention by biofilters under outdoor conditions are presented. The biofilters were filled with a mixture of a soil + peat constituent (25% by volume for each of them) and the rest (50%) with straw or with composted manure ot with chitin (in this later case at the rate of 5 g chitin per liter of substrate). The soil + peat constituent was made either of a material already challenged by pesticides (= recycled biofilters) or of untreated material (new biofilters). Selected pesticides (atrazine, carbofuran, chloridazon, chlortoluron, cyanazine, isoproturon and lenacil) were applied onto biofilters and the eluates were collected and analyzed. Two successive injections of pesticides into the biofilters were conducted. After the first pesticides application, the recycled biofilters made of soil + peat previously treated with pesticides had better retention and degradation rates than the new biofilters. Adding manure also improved these properties of biofilters. Columns made of unchallenged soil + peat and straw (new biofilters) were the least satisfactory: up to 25% of carbofuran were lost. Biofilters made of unchallenged soil + peat and chitin retained the least lenacil. Atrazine was the most retained by biofilters (either new or recycled) with added chitin. Cyanazine was almost absent in the percolates of all biofilters. After the second application of carbofuran and isoproturon, all biofilters improved to the point where (with the exception the new biofilters made of chitin) they retained the totality of the pesticides.

Sequential elution of denatured proteins, hydrolyzed RNA, and plasmid DNA of bacterial lysates adsorbed onto stacked DEAE-cellulose membranes.

Modified membrane chromatography is emerging as a widely applicable technique for the separation of macromolecules. The use of DEAE-cellulose membranes (MemSep) for the purification of bacterial plasmid DNA has been assessed. Cleared bacterial lysates were injected directly onto the membranes without any prior sample cleanup. A single phenol extraction of adsorbed DNA was carried out and about 60 times less phenol was required to achieve the same extent of deproteination for DNA adsorbed onto the membranes as compared to DNA in solution. After chloroform and ethanol wash, (oligo)ribonucleotides resulting from RNase treatment were desorbed with 0.3 M LiCl, 5 mM LiOH. Finally, DNA was eluted with 0.5 M NaCl, 5 mM NaOH. The complete elution of DNA required NaOH in addition to NaCl and the latter salt was a better eluent than LiCl. Without RNase treatment, plasmid DNA-RNA complex required 2 M NaCl, 5 mM NaOH to be completely desorbed. The whole procedure took less than 40 min. The DEAE-cellulose membranes can withstand more than 100 cycles of regenerations and uses without any noticeable decrease of their binding capacity. No cross-contamination of successive DNA preparations was observed. Plasmid DNA was a good substrate for DNA endonucleases. Restriction fragments repurified by this procedure were amenable to ligation. Transformation of Escherichia coli and Saccharomyces cerevisiae with plasmid DNA was observed.

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells.

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.

Kinetics of spheroplasts formation from selected yeast strains.

The formation of spheroplasts from several Kluyveromyces and Saccharomyces strains was studied with the Coulter counter technique. When yeasts were incubated with zymolyase, the number of intact cells decreased according to first-order kinetics after an initial lag. In the concentration ranges studied, the rates were proportional to the cell-lytic enzyme concentrations. The rate (-0.099 min-1) of the most susceptible strain, K. fragilis, was 80-fold higher than that of S. cerevisiae CBS 5495. Compared to the cell sizing technique, the spectrophotometric determination leads to the underestimation of the reaction course.