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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.
Genetic variants within the COL5A1 gene are associated with ligament injuries in physically active populations from Australia, South Africa, and Japan
Published in European Journal of Sport Science, 2023
Javier Alvarez-Romero, Mary-Jessica N. Laguette, Kirsten Seale, Macsue Jacques, Sarah Voisin, Danielle Hiam, Julian A. Feller, Oren Tirosh, Eri Miyamoto-Mikami, Hiroshi Kumagai, Naoki Kikuchi, Nobuhiro Kamiya, Noriyuki Fuku, Malcolm Collins, Alison V. September, Nir Eynon
Genomic DNA was extracted from 4.5 ml of venous blood using a rapid non-enzymatic ethanol precipitation as previously described by Lahiri and Nurnberger (Lahiri & Numberger, 1991) with slight modifications (Mokone, Schwellnus, Noakes, & Collins, 2006). The DNA samples were stored at −20°C until further analysis and freeze–thaw cycles were avoided. DNA concentration was measured at A260 and A280 and the DNA quality was checked by agarose gel electrophoresis. Standard polymerase chain reaction (PCR) was performed using a PCR Express Thermal Cycler (Hybaid Limited, Middlesex, UK). The rs12722 variant was genotyped by using the BSTUI restriction fragment length polymorphism and polyacrylamide gel electrophoresis as previously described (Mokone et al., 2006). The rs10628678 variant was genotyped using predesigned TaqMan® SNP Genotyping Assays (Applied Biosystems, USA) on the Applied Biosystems StepOnePlus™ Real-Time PCR System and the genotypes were called using the Applied Biosystems StepOnePlus™ Real-Time PCR software v2.2.2. For quality control purposes, each 96-well PCR plate included repeat samples, positive controls (samples of a known genotype) and negative controls to ensure consistency in genotype calling which were also confirmed by two researchers in the laboratory. Genotype data from COL5A1 rs12722 C > T was incorporated from previous study with consent of authors.
Influence of 2,4-D residues on the soil microbial community and growth of tree species
Published in International Journal of Phytoremediation, 2020
Luciana Monteiro Aguiar, José Barbosa dos Santos, Gabriela Madureira Barroso, Marcelo Luiz de Laia, Janaína Ferreira Gonçalves, Vitor Antunes Martins da Costa, Lílian Almeida Brito
The genetic profile of distinct groups of microorganisms was determined using PCR-Multiplex (Singh et al. 2006). PCR comprised 1X reaction buffer, 2mM MgCl2, 200 lM dNTPs of each deoxynucleotide, primers [200 μM 63f (10 μM), 200 nM 1087R (10 μM), 400 nM ITS1F (20 μM), 400 nM ITS4 (20 μM), 400 nM Ar3f (20 μM), 400 nM Ar927 (20 μM)], 2.5 U Taq polymesare enzyme, 2 μL template DNA and ultra-pure water to complete 50 μL of reaction. The DNA was amplified in a Biorad automatic cycler (MyCycler™ Thermal Cycler System) programed for 5 min followed by 30 cycles of denaturation (30 sec at 95 °C), annealing (1 min at 55 °C) and extension (1 min at 72 °C) and one final extension step (10 min at 72 °C W). The amplified product (2 μL aliquot) was evaluated by 1.5% agarose gel electrophoresis in 1X TAE buffer stained with Etho Bromide and analyzed under UV light. The amplified products were purified with the QIAquick PCR purification kit protocol reagent kit (QIAGEN), according to the manufacturer's guidelines. The purification product was subjected to enzymatic digestion using the restriction enzyme MspI (Promega, Madison, WI, USA). The digestion was conducted in a thermocycler at 37 °C for 3 h, followed by a 15 min enzyme inactivation period at 95 °C.
Gene expression and biochemical response of giant reed under Ni and Cu stress
Published in International Journal of Phytoremediation, 2019
Shahida Shaheen, Rafiq Ahmad, Qaisar Mahmood, Arshid Pervez, Mohammad Maroof Shah, Farhan Hafeez
The coding sequences of concerned genes available at Gene Bank were utilized for the identification of stress-responsive genes in giant reed plant. Volume of PCR reaction mixture was 10 µL which contained 1 µL cDNA, 1 µL pairs of specific primers, 5 µL 2X Master Mix (Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers) and 2 µL ultrapure water. PCR was performed in a thermal cycler “Master cycler gradient” (Applied Biosystems, USA). Optimized conditions for PCR amplification reaction were adjusted as follows: 35 cycles of amplification followed denaturation (94 °C for 5 min), denaturation (94 °C for 30sec), annealing temperatures were primers dependent so for GR (52 °C for 30 sec), carotenoid hydroxylase (52 °C for 30 sec), bHLH (54 °C for 30 sec), amidase (54 °C for 30 sec), NRAMP (60 °C for 30 sec), Ysl (58 °C for 30 sec) and S19 (56 °C for 30 sec), elongation (72 °C for 30 sec) and final extension (72 °C for 10 min). The amplified PCR products of each gene were visualized with 1.5% agarose gel electrophoresis (Tiangen Biotech, Shanghai). Ethidium bromide-stained gels were scanned and analyzed (Bio-Rad, USA).