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A Comprehensive HLA-DRB, -DQB, and -DPB Oligotyping Procedure by Hybridization with Sequence-Specific Oligonucleotide Probes
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
The cycling conditions for all primer combinations are given in Table 1. The samples are heated first at 100°C for 7 min before adding the Taq polymerase. In all PCR series a blank sample is included to monitor for DNA carryover. The efficiency of PCR is checked by electrophoresis in 0.7% agarose gel in TBE buffer.
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
4. DNA Electrophoresis. Horizontal 1.0% agarose (BRL UltraPure) electrophoretic gels were run in 45 mM Tris Borate, 1 mM EDTA, pH 8.3 (0.5X TBE) buffer with recirculation. Gels were stained after running with 1.0 μg/ml ethidium bromide and photographed with a red filter while transilluminated by a 300 nm UV source. Low voltage electrophoresis (LoVE) was performed at room temperature for 24 hours at a field strength of 1.7 V/Cm. All other gels were run at 14°C with a field strength of 5.6 V/cm. FIGE was performed with a forward to reverse pulse ratio of 3.0, with pulses ramped linearly from an initial forward pulse time of 3 seconds to a final forward pulse time of 60 seconds over 12 hours. Linear DNA molecular weights were determined with intact chromosomes of S. cerevisiae (Beckman) and coliphage lambda DNA digested with HINDIII restriction endonuclease (BRL).
Sperm chromatin assessment
Published in David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham, Textbook of Assisted Reproductive Techniques, 2017
Ashok Agarwal, Rakesh Sharma, Gulfam Ahmad
In the two-tailed comet technique, sperm cells are diluted in PBS to a concentration of 10 x 106 spermatozoa/mL. A 25-μL cell suspension is mixed with 50 μL of 1% low-melting point agarose in distilled water at 37°C. A total of 15 μL of the mixture is placed on the slide, covered with a coverslip, and transferred to an ice-cold plate. As soon as the gel solidifies, the coverslips are removed and the slides are rinsed in two lysing solutions: lysing solution 1 (0.4 mol/L Tris-HCl, 0.8 mol/L DTT, and 1% SDS, pH 7.5) for 30 minutes, followed by lysing solution 2 (0.4 mol/L Tris-HCl, 2 mol/L NaCl, 1% SDS, and 0.05 mol/L EDTA, pH 7.5) for 30 minutes. Then, the slides are rinsed in TBE buffer (0.09 mol/L Tris-borate and 0.002 mol/L EDTA, pH 7.5) for 10 minutes, transferred to an electrophoresis tank, and immersed in fresh TBE electrophoresis buffer. Electrophoresis is performed at 20 V (1 V/cm) and 12 mA for 12.5 minutes. After washing in 0.9% NaCl, nucleoids are unwound in an alkaline solution (0.03 mol/L NaOH and 1 mol/L NaCl) for 2.5 minutes, transferred to an electrophoresis chamber, and oriented at 90° to the first electrophoresis.
Low dose gamma irradiation pretreatment modulates the sensitivity of CNS to subsequent mixed gamma and neutron irradiation of the mouse head
Published in International Journal of Radiation Biology, 2021
Alla V. Rodina, Yulia P. Semochkina, Olga V. Vysotskaya, Anastasia N. Romantsova, Aleksandr N. Strepetov, Elizaveta Y. Moskaleva
Reverse transcription of 1 μg of RNA was performed with Oligo (dT) 17-primers from the MMLV RT kit (Evrogen) in 20 μL of the reaction mixture. PCR was carried out in a reaction mixture (qPCRmix-HS, Evrogen) with a final volume of 25 μL with the addition of 7 pmol of each of the primer pairs and 3 μL of an RT reaction mixture containing cDNA. Temperature-time parameters of PCR were as follows: 94°С – 30 s (first cycle – 3 min), 58°С – 30 s (first cycle – 2 min), 72°С – 1 min (first cycle – 3 min, last cycle – 7 min), the number of cycles for neurotrophins is 35 cycles, for β-actin – 28 cycles. The gene primer sequences for RT-PCR are listed in Table 2. RT-PCR products were separated by electrophoresis in 1.5% agarose gel containing 0.5 μg/mL ethidium bromide. TBE buffer (0.1 M Tris, 0.09 M boric acid, 1 mM EDTA, pH 8.3) was used as a buffer for electrophoresis. The samples were placed in the wells of the gel in a volume of 8 μL. The results were visualized using the Syngene G: BOX Chemi XT4 gel documentation system (Great Britain) with the built-in GeneTools image processing program. The expression levels of the studied genes were normalized to the expression level of the constitutive β-actin gene. Each experiment had been repeated 3 times.
DNA-dependent protein kinase: effect on DSB repair, G2/M checkpoint and mode of cell death in NSCLC cell lines
Published in International Journal of Radiation Biology, 2019
Ali Sak, Michael Groneberg, Martin Stuschke
Uniformly labeled cells (1.85 kBq [2-14C]thymidine/ml (1.92 MBq/mmol) for 72 h were sub-cultured to low density of 0.5–1 × 105 cells/cm2 and were irradiated 20 h later with 30 Gy. Cells were sampled at 0 and 4 h post irradiation and were cast into plug moulds. Plugs were transferred into lysis buffer (100 mM EDTA, 10 mM Tris, 20 mM NaCl, 1 mg/ml proteinase K, 1% (w/v) sodium lauryl sarcosine, pH 8.0) and incubated for 24 h at 50 °C. Plugs were then washed and cut into 5-mm pieces containing ∼1 µg DNA. For electrophoresis, plugs were loaded on wells of 0.75% agarose. Electrophoresis was carried out in 0.5× TBE buffer (45 mM Tris borate, 1 mM EDTA, pH 8.0) at room temperature for 72 h at a constant field strength of 0.6 V/cm. After staining with ethidium bromide the wells and each lane of the gel were cut into separate segments and the radioactivity was determined. The fraction of radioactivity released from the plugs into the gels (FAR) was calculated according to:
Poly(glycerol methacrylate)-based degradable nanoparticles for delivery of small interfering RNA
Published in Pharmaceutical Development and Technology, 2018
Noha G. Morsi, Shimaa M. Ali, Sherouk S. Elsonbaty, Ahmed A. Afifi, Mostafa A. Hamad, Hui Gao, Mahmoud Elsabahy
Low molecular weight chitosan (50,000–190,000 Da), cyanine 5-labled siRNA and polyethylenimine (branched PEI, average molecular weight of 60,000 Da) were purchased from Sigma-Aldrich (St. Louis, MO). Ultra-pure water (free from RNAse and DNAse) was purchased from Biochrom GmbH (Berlin, Germany). Tris buffer was purchased from Fisher Scientific (Waltham, MA). Ethidium bromide and phosphate-buffered saline (PBS) were purchased from MP Biomedicals (Santa Ana, CA). Tris–borate–EDTA (TBE) buffer and agarose (molecular biology grade) were purchased from Fischer Scientific. Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Lonza (Basel, Switzerland). Fetal bovine serum (FBS) was purchased from Biowest (Riverside, MO). Cell titer 96® Non-Rad cell proliferation assay (MTT) was purchased from Promega (Madison, WI). Trehalose was purchased from LOBA Chemie (Mumbai, India). All other reagents were obtained from Sigma Aldrich (Shanghai, China) and used as received, except for tetrahydrofuran (THF), which was dried by refluxing over sodium, in the presence of benzophenone as an indicator.