Purification of Labeled Oligonucleotides by Precipitation with Ethanol

If labeled oligonucleotides are to be used only as probes in hybridization experiments, complete removal of unincorporated label is generally not necessary. However, to reduce background to a minimum, the bulk of the unincorporated label should be separated from the labeled oligonucleotide. Most of the residual unincorporated precursors can be removed from the preparation by differential precipitation with ethanol, as described in this protocol, if the oligonucleotide is >18 nt in length.

Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography

When labeled oligonucleotides are to be used in enzymatic reactions such as primer extension, virtually all of the unincorporated label must be removed from the oligonucleotide. For this purpose, chromatographic methods or gel electrophoresis are superior to differential precipitation of the oligonucleotide with ethanol or cetylpyridinium bromide (CPB). This protocol describes a method to separate labeled oligonucleotides from unincorporated label that takes advantage of differences in mobility between oligonucleotides and mononucleotides during size-exclusion chromatography. Although size-exclusion chromatography can, in principle, be used to purify either radiolabeled or nonradiolabeled oligonucleotides, this protocol is geared toward purifying radiolabeled oligonucleotides, whose elution from the column is monitored using a minimonitor and whose separation from unincorporated nucleotides is monitored by liquid scintillation counting.

UV Detection and Avoidance of Protein in Basella alba leaf Mucilage Polysaccharide by differential precipitation

Objective: In the aim of refining mucilage polysaccharide extracted from the leaf of Basella alba, extraction and differential precipitation of the protein content was studied. This was attempted by pH adjustment by adding Trichloroacetic acid (TCA) and Hydrochloric acid (HCl). Materials and Methods: The presence of residual protein before and after deproteinization in the polysaccharide solution was detected by UV spectrum analysis. The % polysaccharide, % polysaccharide loss, protein concentration and the deproteinization efficiency were studied as comparative indices to evaluate the precipitation experimental conditions using pH adjustment. Results: The result showed that 10% w/v TCA precipitated over 80% of the protein when the pH of the aqueous polysaccharide solution was 3. Discussion: TCA was proved to be superior to hydrochloric acid as evidenced by the highest deproteinization efficiency (83.3%). The polysaccharides of all the extracted solutions obtained were identified with only slight variations in percentage. The Hcl method excelled over the TCA method in obtaining polysaccharides with little lower percentage of polysaccharide loss (13.94 %). Conclusion: The TCA method will offer a room for deproteinization of polysaccharides if optimization is studied.

Differential Precipitation of Mg(OH)2 from CaSO4·2H2O Using Citrate as Inhibitor—A Promising Concept for Reagent Recovery from MgSO4 Waste Streams

In hydrometallurgical processing and acidic wastewater treatment, one of the neutralizing agents employed is MgO or Mg(OH)2. At the end of this process, the resulting solution, which is rich in SO42− and Mg2+ is treated with lime to remove (or minimize the amount) of these ions via the precipitation of Mg(OH)2 and CaSO4·2H2O (gypsum). In our work, an attempt was made to separate the two solids by increasing the induction time of the gypsum precipitation, thus regenerating relatively pure Mg(OH)2 which could be reused in wastewater treatments or hydrometallurgical processing circuits, and in this way, significantly enhancing the economic viability of the process. During our experiments, the reaction of an MgSO4 solution with milk of lime prepared from quicklime was studied. The effects of a range of organic additives, which can slow down the precipitation of gypsum have been assessed. The process was optimized for the most promising inhibiting agent—that is, the citrate ion. The reactions were continuously monitored in situ by conductometric measurements with parallel monitoring of solution pH and temperature. ICP-OES measurements were also carried out on samples taken from the reaction slurry. The composition of the precipitating solids at different reaction times was established by powder XRD and their morphology by SEM. Finally, experiments were carried out to locate the additive after the completion of the precipitation reaction to get information about its potential reuse.

TRANSCRIPTION ANALYSIS OF GENE EXPRESSION ISLAND VcB V. CHOLERAE BY METHOD OF FULL-GENOMIC SEQUENCING

The aim of the study was to analyze the expression of V. cholerae genes that are part of the VcB island by means of full-genomic sequencing of the transcriptome. The VcB island is localized on the second chromosome in all toxigenic vibrios studied and is absent in the atoxigenic apiliated strains. Two strains of V. cholerae O1 ctxA+ tcpA+ and one strain V. cholerae O1 ctxA– tcpA– were studied. The pool of total RNA vibrios was isolated by a technique based on differential precipitation in the presence of lithium ions. In the total pool of sequenced RNA, RNA encoded in the order of 3 500 by known cholera vibrio genes was identified. In a pool of total RNA from two ctx + tcpA + strains RNA transcripts were found for the five genes included in the VcB island, excluding the VCA0282 gene, previously identified as the ISVch5-transposase. In the the pool of total RNA from the ctx– tcpA– strain no transcripts of the two genes previously described as VCA0282-transposase and VCA0283 were detected. A possible explanation may be the existence of copies of these genes in other parts of the genome of the ctxA– tcpA– V. cholerae.