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Laboratory tutorials
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2018
Single-strand conformation polymorphism (SSCP) technique is a simple and efficient means to detect any small alteration in PCR-amplified products. It is based on the assumption that subtle nucleic acid changes affect the migration of single-stranded DNA fragments and, therefore, results in visible mobility shifts across a nondenaturing PAGE. PAGEs were used for analysis of DNA with specialized buffer systems and without urea. In nondenaturing PAGE, the components used to synthesize matrix were acrylamide monomers, N,N-methylenebisacrylamide (Bis), ammonium persulfate (APS) and TEMED. APS when dissolved in water generates free radicals, which activate acrylamide monomers, inducing them to react with other acrylamide molecules, forming long chains. These chains cross-linked with Bis. TEMED acts as a catalyst for gel formation because of its ability to exist in free radical form. The acrylamide and bisacrylamide was used in a 49:1 ratio, adding autoclaved HPLC water to make 100 mL volume. This 49:1 acrylamide–bisacrylamide solution was dissolved completely using a magnetic stirrer and kept in refrigerator until required.
Glossary of scientific and technical terms in bioengineering and biological engineering
Published in Megh R. Goyal, Scientific and Technical Terms in Bioengineering and Biological Engineering, 2018
Single strand conformational polymorphism (SSCP) is a technique for detection of mutations in a defined DNA sequence. Single-stranded polynucleotides are electrophoretically separated on non-denaturing gels. Intrachain base pairing results in a limited number of conformers stabilized by intrachain loops, and mutated DNA shows on electrophoresis an altered assortment of such conformers.
Long-term corrosion behaviour of carbon steel and stainless steel in Opalinus clay: influence of stepwise temperature increase
Published in Corrosion Engineering, Science and Technology, 2019
Sophia Necib, Michel L. Schlegel, Christian Bataillon, Sylvie Daumas, Nikitas Diomidis, Peter Keech, Didier Crusset
Biomolecular and phylogenetic analyses were performed on one sample of the water from the inner gap following a standard approach [6]. Typically, DNA extraction was followed by 16s rRNA amplification (V3 fragment) by Polymerase Chain Reaction (PCR). Quantification of the total prokaryote content was performed by quantitative PCR (qPCR) using UV-based DNA quantification (NanoQuant technique) and Fluorescence-based DNA quantification (Qubit technique). The sample amplicons were also analysed by two complementary methods. For the Single Strand Conformation Polymorphism (SSCP) method, DNA was denatured as single strands and separated by capillary electrophoresis using an automatic sequencer. Each SSCP electrophoregramm peak then corresponds to the contribution of at least one species and the peak area reveals the species relative abundance. For the Denaturing Gradient Gel Electrophoresis (DGGE) method, DNA double strands were separated on a polyacrylamide gel. The major and minor DGGE bands were later excised and amplified, then sequenced for phylogenetic analyses.