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Security Vulnerabilities of Quantitative-Analysis Frameworks
Published in Mohamed Ibrahim, Krishnendu Chakrabarty, Optimization of Trustworthy Biomolecular Quantitative Analysis Using Cyber-Physical Microfluidic Platforms, 2020
Mohamed Ibrahim, Krishnendu Chakrabarty
Having filled in all the tubes on the PCR plate, the tubes were closed, then placed in a balanced micro-centrifuge, and spun for a few seconds. Immediately after the spin, we placed the reaction tubes in the thermal cycler, which had been programmed to perform DNA amplification by continuously raising and lowering the temperature of the PCR-plate content in discrete, pre-programmed steps. Figure 9.12 shows the transfer of PCR samples into the thermal cycler. After the PCR program had finished, we transferred the PCR-plate contents into the gel electrophoresis apparatus, allowing the chemical solutions to migrate over the gel with the aid of an electric field; Figure 9.13 shows the transfer of 8 × 4 samples into the gel. Note that the gel was virtually divided into four sections, and each section was loaded with samples from a certain row of tubes on the PCR plates. This approach allowed us to easily compare the outcomes of all the PCR runs. Also, in each section, we pipetted a specific chemical reagent, known as DNA ladder, which has fragments of DNA of different sizes, allowing us to benchmark the results of the PCR-related chemical solutions. Gel electrophoresis was run for an hour, then the gel block was transferred to a chamber that contains a source of UV light in order to visualize the DNA bands. Figure 9.14 shows a view of the gel after the electrophoresis process, viewing the migration of PCR samples. Result interpretation is discussed next.
Innovations in Noninvasive Instrumentation and Measurements
Published in Robert B. Northrop, Non-Invasive Instrumentation and Measurement in Medical Diagnosis, 2017
The PCR procedure consists of the following operations: Initialization step: (only required for DNA polymerases that require heat activation by hot-start PCR). The reactants are heated for 1–9 min to 94–98°C.Denaturation step: This step is the first regular thermal cycling step, and consists of heating the reaction to 94–98°C for 20–30 s. It causes separation of the DNA helix by breaking the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.Annealing step: The reaction temperature is lowered to 50–65°C for 20–40 s, allowing annealing of the primers to the single-stranded DNA template. This temperature needs to be low enough to allow for hybridization of the primer to the DNA strand, but high enough for the hybridization to be specific, that is, the primer should only bind to a perfectly complementary part of the template. The polymerase binds to the primer–template hybrid molecule and begins DNA formation.Extension–elongation step: In this step, the reaction temperature is made 72–80°C, an optimum for the DNA polymerase (Taq polymerase) to function. In this step, the DNA polymerase assembles a new DNA strand complementary to the DNA template by adding dNTPs that are complementary to the template in its 5′–3′ direction, condensing the 5′ phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the growing DNA strand. The growth time depends both on the DNA polymerase used and the length of the target DNA fragment being duplicated. As a rule of thumb, at the optimum temperature, the DNA polymerase will add 1000 bases per minute. Under optimum (ideal) conditions, at each extension step, the amount of DNA target is doubled, leading to exponential amplification of the specific DNA target fragment.Final elongation: This single step occasionally may be performed at a temperature of 70–74°C (this is the optimal temperature for most polymerases used in PCR) for 5–15 min after the last PCR cycle to ensure that any remaining ssDNA is fully extended.Final hold: This step is held at 4–15°C for an indefinite time and is used for short-term storage of the reaction product.Check: To check whether the PCR process generated the anticipated DNA fragment (also known as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of the products is determined by comparison with a DNA “ladder” (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products.
Isolation and identification of new strains of crude oil degrading bacteria from Kharg Island, Iran
Published in Petroleum Science and Technology, 2018
Kazem Godini, Mohamad Reza Samarghandi, Doustmorad Zafari, Ali Reza Rahmani, Abbas Afkhami, Mohammad Reza Arabestani
In order to confirm the extracted isolates that were detected by phenotypic methods, PCR assay was applied and the reaction was done using Bio-Rad Thermal Cycler; thus, so as to amplify the 16S rRNA gene using an universal bacterial primer 8F (forward-5' GGATTAGATACCCTGGTAGTCC-3' and reverse-5'-TCGTTGCGGGACTTAACCCAAC-3'). The PCR mix contained 1 µL (10 pmol) of the primer, 2 µL DNA, 12.5 µL PCR Master Mix in a final 25 µL reaction volume. DNA amplification was conducted in a thermal cycler (S1000™ Thermal Cycler, Bio-Rad, Hercules, CA, USA), under the following conditions: initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 1 min, an annealing temperature at 57 °C for 1 min, an extension at 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. Electrophoresis of the amplified DNA fragments, along with a 100bp DNA ladder, was carried out using 2% agarose gel (Merck, Darmstadt,- Germany).
Direct and cost-effective method for histone isolation from cultured mammalian cells
Published in Preparative Biochemistry & Biotechnology, 2023
Anja Batel, Mirjana Polović, Mateo Glumac, Andrea Gelemanović, Matilda Šprung, Ivana Marinović Terzić
Standard 2% agarose gels were prepared by dissolving 1 g of agarose powder (UltraPure™ agarose 16500 Invitrogen) in 50 mL of 1X TAE buffer (40 mM Tris, 2 mM EDTA, 20 mM acetic acid, pH 8). Ethidium bromide (0.2 µg/mL) was added to dissolved agarose. Samples, fractions collected from the protocol, were mixed with a DNA loading dye (2.5 mg/mL bromophenol blue (Sigma B5525), 2.5 mg/mL xylene cyanol (Sigma X4216), 30% glycerol). GeneRuler 100 bp DNA Ladder (Thermo Scientific SM0241) was used as a reference size ranging from 100 to 1000 bp. Gels were run at 100 V for 1 h. DNA fragments were visualized by the Uvidoc HD6 imaging system (UVITEC Cambridge).