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Basic Molecular Cloning of DNA and RNA
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
Single-stranded RNA can be manipulated in a similar fashion to double-stranded DNA, with certain exceptions. The most important exception is that, while enzymes that degrade DNA (DNases) are heat labile and require divalent cations, RNases cannot be destroyed by autoclaving, require no cofactors, and are ubiquitous in the environment. (All organisms produce them; common sources in the laboratory are bacteria and culture media, and human sweat, saliva, and tears.) RNA work thus requires dedicated pipettes and tips, specially prepared solutions, and meticulous technique. Resources are available from molecular biology suppliers to aid in RNase decontamination. One of the most common is to add diethyl pyrocarbonate (DEPC) to any solutions used for RNA work. DEPC covalently modifies histidines and deactivates nucleases. (Note that it should not be used with amine-containing buffers, such as Tris.) However, it is toxic and can inhibit some downstream applications, so final purified RNA should not be dissolved in DEPC water. Additional products with names such as RNaseZap and RNase AWAY are also sold for cleaning benchtops and glassware or plasticware.
Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Diethyl pyrocarbonate (DEPC) reacts with the amines of proteins, such as RNAses and DNAses, rendering them inert. Following autoclaving, DEPC degrades harmlessly, leaving an RNAase- and DNAse-free solution. However, due to the reaction with amines, DEPC should not be used to treat amine-containing buffers such as Tris.
Novel attacin from Hermetia illucens: cDNA cloning, characterization, and antibacterial properties
Published in Preparative Biochemistry and Biotechnology, 2019
To prepare the cDNA library, total RNA was isolated from the induced fat bodies of H. illucens using Trizol (Invitrogen), according to the manufacturer’s protocol and was dissolved in water treated with diethyl pyrocarbonate. The full-length sequence of the H. illucens attacin (HI-attacin) gene was revealed by RACE. The first-strand cDNA was synthesized from 1 µg of total RNA using a SMARTer RACE cDNA Amplification Kit (Clontech, USA). For 3′-RACE, a DNA fragment encoding a portion of HI-attacin was amplified by PCR using the Advantage 2 PCR Kit (Clontech) with forward primers (Table 1) and a universal primer mix (UPM) for the reverse primers. The 3′-RACE forward primer was designed based on high degree of homology in the amino acid sequences of dipteran attacins. The RACE-PCR product was cloned into the TA topo vector (Doctor protein) and the nucleotide sequence was determined from both the ends using an ABI-3700 automatic DNA sequencer (Applied Biosystems, USA). For 5′-RACE, a gene-specific primer (Table 1) was designed from the internal sequence obtained from the previous 3′-RACE-PCR. The nucleotide sequencing was conducted as described above. The sequence alignment was carried out using the Clustal W multiple sequence alignment program and phylogenetic tree was generated using the MEGA 6 program.
Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS
Published in Preparative Biochemistry & Biotechnology, 2018
Yogesh Devaraj, Savita Kumari Rajender, Prakash Motiram Halami
Characterization of the enzyme was performed using azocasein, a chromogenic plasmin substrate.[28] The purified enzyme was incubated with 100 (l of azocasein (3 mg/ml) dissolved in distilled water and the assay was performed according protocol followed by Ambrose et al.[29] To study the effect of pH on CFR15-protease activity, buffers with strength of 10 mM, and in the range 2.5–12 were used. The buffer systems used were as follows, HCl (pH 2.5), sodium acetate buffer (pH 3.5–5.5), phosphate buffer (pH 6.5–8.5), carbonate buffer (pH 10.5–11), and NaOH (pH 12). The effect of temperature was studied at various temperatures ranging from 35 to 70°C. Effects of various metal ions were studied at 10 mM concentration. Metal chlorides of Na+, Mn2+, K+, Mg2+, Cu2+, Ba2+, Cs2+, and Ca2+ were used for the source of metal ions for the study and the protease activity was studied at 37°C for 30 min. To study inhibitory action of various protease inhibitors, the enzyme was pre-incubated for 30 min with iodo acetic acid, phenyl methyl sulphonyl fluoride (PMSF), dithiothreitol, ethylene diamine tetra acetic acid (EDTA), diethyl pyrocarbonate, Pepstatin-A at the concentration of 10 mM and the residual activity was estimated.