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Optimal Detection of and Effect of Vitamin D3 on Extrachromosomal Oncogene Sequences
Published in Maryce M. Jacobs, Vitamins and Minerals in the Prevention and Treatment of Cancer, 2018
Daniel D. Von Hoff, Donald R. VanDevanter
2. Preparation and Irradiation of Cells in Blocks. Cells were harvested, rinsed with PBS, and suspended in PBS at a density of 2.5 × 107 cells/ml. Cell suspensions were mixed with an equal volume of molten (55°C) 1% low melting point agarose (Sea-Plaque, FMC Corporation, Rockland, ME) in PBS and pipetted in 40-μl aliquots (5 × 105 cells) into a rubber-based block-forming mold.23 Blocks were allowed to harden and were placed in 15-ml culture tubes containing 2 ml of 1% Sarkosyl (Sigma Chemical Co.), 100 mM EDTA. Proteinase K (2 mg/ml) was added, the blocks were incubated for 24 h at 55°C, rinsed twice with 10 ml of TE, and stored in TE at 4°C. Blocks were placed in 100 μl of TE in 1.5 ml microfuge tubes, and irradiated by 137Cs source (Gamma Cell 40, Atomic Energy of Canada Ltd., Ottawa, Canada) at a dose of 1.2 Gy/min for 6 min before electrophoresis.
Demonstration of the Mutation Associated with Porcine Stress Syndrome
Published in S. Tsuyoshi Ohnishi, Tomoko Ohnishi, Malignant Hyperthermia, 1994
Xia Zhang, Hua Shen, C. Robert Cory, Peter J. O’Brien
20 mg/ml Proteinase K stock 20 mg of Proteinase K (Boehringer Mannheim Canada Ltd., Laval Que) in 1.0 ml of H2O, stored at -85°C. The solution is mixed by gentle inversion and aliquoted in 200-μl volumes into microcentrifuge tubes. These aliquots may undergo several freeze thaw cycles with minimal losses in activity.
Characterization of the 2 M NaCl-Resistant Chromatin Fraction from Chicken Erythroid Cells
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
Peter C. Hentzen, Isaae Bekhor
All DNA samples were made 1% in sodium dodecyl sulfate (SDS) to which 50 μg/ml proteinase K was added, and incubated for 2 hr at 37°C. The DNA was extracted twice with an equal volume of chloroform to isopentyl alcohol (24:1) for 20 min and the phases resolved by centrifugation. The aqueous phase was made 0.3 M in NaCl, and 2.5 volumes of cold ethanol was added to precipitate the DNA at — 20°C. The precipitated DNA was dissolved in distilled water, 20 μg/ml RNase A was added, and it was incubated for 2 hr at 37°C. Proteinase K (50 μg/ml) and SDS (1%) were later added and incubation continued for 2 hr at 37°C. The DNA solution was extracted twice with an equal volume of chloroform to isopentyl alcohol as above, and once with an equal volume of phenol to chloroform to isopentyl alcohol (25:24:1) and resolved by centrifugation. The DNA was precipitated from the final aqueous phase as above. The precipitated DNA was dissolved in distilled water and dialyzed against 4 l of 0.3 M NaCl, 10 mM Tris-HCl, pH 8.0. The purified DNA(A260/A230 = 2.4, A260/A280 = 1.8 to 1.9) was stored at -70°C.
Pre-implantation genetic testing for Marfan syndrome using mini-sequencing
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Krit Makonkawkeyoon, Vorasuk Shotelersuk, Opas Sreshthaputra, Tawiwan Pantasri, Rekwan Sittiwangkul, Theera Tongsong, Wirawit Piyamongkol
The biopsied trophectoderm were washed a minimum of four times with fresh phosphate-buffered saline (PBS, Cell Signaling Technology, Theera Trading Co. Ltd., Bangkok, Thailand) droplets with 0.1% polyvinyl alcohol (PVA, Sigma-Aldrich, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) before transferring to 200 µL microcentrifuge tubes with a total volume of 2 μL. A 2 μL aliquot of the last washing drop PBS was also taken as a control for each embryo. Lysis buffer of 1 μL of 17 mmol/L sodium dodecyl sulphate (SDS, Sigma®, St. Louis, MO) and 2 μL of 125 mg/mL proteinase K (PK, Roche Diagnostics (Thailand) Ltd., Bangkok, Thailand) was added into each tube. Samples were then incubated at 37 °C for 1 h, proteinase K was then inactivated by heating at 99 °C for 15 min on a thermal cycler (Roche Diagnostics (Thailand) Ltd., Bangkok, Thailand) (Piyamongkol et al. 2012).
Inhibitory effect of proteinase K against dermatophyte biofilms: an alternative for increasing the antifungal effects of terbinafine and griseofulvin
Published in Biofouling, 2022
Raimunda Sâmia Nogueira Brilhante, Raissa Geovanna Pereira Lopes, Lara de Aguiar, Jonathas Sales de Oliveira, Géssica dos Santos Araújo, Germana Costa Paixão, Waldemiro de Aquino Pereira-Neto, Rosemayre Souza Freire, João Victor Serra Nunes, Renan Pereira de Lima, Flávia Almeida Santos, José Júlio Costa Sidrim, Marcos Fábio Gadelha Rocha
The dermatophyte biofilms treated with proteinase K (32 µg ml−1) showed a reduction in biomass (38%). In a study by Al-Fattani and Douglas (2006), biofilms of C. albicans and C. tropicalis treated with proteinase K (50 µg ml−1) showed biomass reductions of 7% and 31%, respectively. On the other hand, treatment of biofilm of Staphylococcus aureus with 100 μg ml−1 of PK reduced their mass by 14–75% (Dakheel et al. 2016). In a study of clinical strains of Staphylococcus aureus resistant or susceptible to methicillin, proteinase K reduced the mature bacterial biofilm by up to 76.5% at concentrations higher than those used in the present study (100 µg ml−1) (Sugimoto et al. 2018). Proteinase K has enzymatic action and targets a broad range of peptide bonds in proteins, one of the main constituents of the extracellular matrix of biofilms (Shukla and Rao 2017; Hu et al. 2020). These differences in proteinase K activity against biofilms from different microbial species may be partially explained by the fungal biofilm composition (Windham et al. 2018). Proteins are not the only component that helps the maintenance and protection of biofilms, despite being an important component of the extracellular matrix, and their levels being different among biofilms of different species.
Diagnosis of pleural empyema/parapneumonic effusion by next-generation sequencing
Published in Infectious Diseases, 2021
Yoshiki Shiraishi, Kirill Kryukov, Katsuyoshi Tomomatsu, Fumio Sakamaki, Shigeaki Inoue, So Nakagawa, Tadashi Imanishi, Koichiro Asano
The pleural fluid samples (obtained by thoracentesis) were fractionated into cellular components and supernatant by centrifugation at 50 × g at 4 °C for 15 min. The supernatants were divided into 1 mL aliquots and stored at −80 °C. Ice-cold aliquots of the supernatants (1 mL) were centrifuged at 20,000 × g at 4 °C for 15 min, and DNA was extracted from the pellets using the DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany). The pellets were resuspended in 200 µL of buffer (10 mM Tris-HCl, 0.1 M NaCl, 1 mM EDTA, 5% (v/v) Triton X-100, pH 8.0) with 20 mg/mL lysozyme and 4 µL of RNase (100 ng/mL) and incubated at 37 °C for 30 min. Proteinase K (25 µL, 20 mg/mL) was added and incubated at 56 °C from 30 min to overnight. Following alcohol precipitation, samples were dissolved in 20 µL of nuclease-free water. DNA was quantified using a Qubit™ fluorometer with a dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA).