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Approaches to the Measurement of Biological Pollutants
Published in Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus, Environmental Chemical Analysis, 2018
Somenath Mitra, Pradyot Patnaik, Barbara B. Kebbekus
The methods of RNA isolation depends upon the tissue and the type of RNA to be extracted. Procedures to isolate total cellular RNA include chemical extractions and centrifugation. Phenol extraction was one of the first techniques to successfully isolate RNA from many sources. However, guanidinium salts have been found to be better options, even for those tissues that are rich in RNA degrading enzymes. Guanidinium hydrochloride and guanidinium thiocyanates are very powerful chaotropic agents. Guanidinium thiocyanate-based methods are quite popular for the isolation of good-quality RNA from a variety of tissues. Cells (or tissues) are homogenized directly in a solution containing guanidinium salt and reducing agents such as 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) to break intramolecular protein disulfide bonds. These conditions rapidly inactivate RNases by distorting the secondary and tertiary folding of the enzymes when the cells are disrupted. Using these reagents, it is possible to isolate intact RNA even from RNase-rich tissues and cells.
Extraction of phenolic pollutants from industrial wastewater using a bulk ionic liquid membrane technique
Published in Environmental Technology, 2022
Khalid Farhod Chasib, Anwer Jassim Mohsen, K. J Jisha, Ramesh L. Gardas
These findings agree with the results of Vidal et al. [58], Fan et al. [39], Khachatryan et al. [59] and Y.S. Ng et al. [4]. According to Fan et al. [39] and Hanke et al. [60], the phenol extraction was reliant on the hydrogen bonding formation between the ILs anion the with molecular phenol. Based on this result, hydrogen-bonding interactions of [NTf2]−and [PF6]− with the hydroxyl (OH) of the phenols is expected. Thus, when the pH of the feed phase is increased, hydrogen bonding becomes weaker. The extraction efficiency of phenol using BMILs is higher than SILs because of the formation of hydrogen bonding between phenol and the two anions [NTf2] and [PF6]. Figure 8 shows that the efficiency of stripping is less affected by the pH of the feed phase.
Retrieving high-quality genomic DNA from formalin-fixed paraffin-embedded tissues for multiple molecular analyses
Published in Preparative Biochemistry & Biotechnology, 2022
Ha Thi Nguyen, Vinay Bharadwaj Tatipamula, Duy Ngoc Do, Thien Chi Huynh, Mai Kim Dang
DNA extraction was performed either with our phenol/chloroform-based protocol or the QIAamp DNA FFPE Tissue kit. If RNA-free DNA is required, RNase (a final concentration of 1 μg/μl) is added to the cell lysate and incubated for 15 min at 37 °C. For phenol/chloroform-based extraction, an equal volume of tris-equilibrated phenol solution (pH = 8.0) (Sigma, St. Louis, MO, No. P 4557) was added and vortexed vigorously, followed by spinning for 5 min at 8000g. The aqueous phase was carefully recovered to a newly labeled 1.5 ml tube (avoiding the milky white material present in the interphase). This phenol extraction step was repeated one more time for the newly obtained aqueous phase to avoid protein contamination. An equal volume of chloroform:isopropanol mixture (24:1, v/v) was added and vortexed vigorously, followed by spinning for 5 min at 8000g in a microcentrifuge. The aqueous layer was carefully retrieved to a new-labeled 1.5 ml tube. Estimating the volume of the aqueous layer that was recovered, 1/10 volume of 3 M sodium acetate pH 5.2 and ∼2 volumes of absolute ice-cold ethanol were added and thoroughly mixed. The tube was kept at −80 °C for at least 20 min or at −20 °C for some hours or overnight, followed by centrifugation at maximum speed, 4 °C for 15 min. The supernatant was carefully discarded to avoid losing the pellet (may be invisible). After that, the pellet was washed with ice-cold ethanol 70% to remove undesirable salts and air-dried at RT or 37 °C. The pellet was then re-suspended in ddH2O or TE buffer.
Assays and enumeration of bioaerosols-traditional approaches to modern practices
Published in Aerosol Science and Technology, 2020
Maria D. King, Ronald E. Lacey, Hyoungmook Pak, Andrew Fearing, Gabriela Ramos, Tatiana Baig, Brooke Smith, Alexandra Koustova
DNA sample preparation used for PCR amplification should be optimized for the successful amplification of increasingly longer targets from genomic DNA (Cheng et al. 1995). Therefore, before the PCR process, the intactness of the genomic DNA should be maintained during the collection and isolation process to create reproducible amplification of fragments with sizes >1.3 kb (Deagle, Eveson, and Jarman 2006). A larger target size can increase the probability of producing an unusable DNA template strand by randomly introducing a single-stranded (ss) nick or double-stranded (ds) break within the target sequence (Zhou, Pape, and Schwartz 2008). The DNA sample preparation varies for each experiment. Genomic DNA is isolated from the sample’s cultured cells by various methods such as boiling in the presence of a chelating resin, using alkaline lysis, or phenol extraction (Cheng et al. 1995). A standard reaction mixture contains the DNA template along with the specified forward and reverse primers, deoxyribonucleotide triphosphate (dNTP) mix, buffer with Mg++ for the specific DNA polymerase, and DNA polymerase. The PCR process typically utilizes thermal cycling, which consists of an initial denaturing step and a series of 20 to 40 cycles of denaturing, annealing and extension steps (Svabenska 2012; Liang and Johnson 1988; Sambrook, Fritsch, and Maniatis 1989).