Suspension of human glioblastoma cells (3 × 105 cells) was incubated in 1 mL DMEM supplemented with 20 µg/mL of mitomycin C (MMC) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 1 h. After incubation, cells were sedimented, washed with medium supplemented with 10% serum, and resuspended in the same medium and seeded on a 24-well plate. Every 6 h, cells were sampled and fixed in agarose blocks (Low Melt Ultra-Pure DNA Grade Agarose; Bio-Rad, Hercules, CA, USA) at 4 °C. After solidification, blocks were transferred into 500 mM EDTA. Prior to electrophoresis, blocks were washed in 1 × TE, transferred into lysis buffer [1% N-lauroylsarcosine (Biophoretics, Sparks, NV, USA), 50 mM EDTA, 1 mg/mL proteinase K] and lysed at 50 °C for 15 min. Electrophoresis was conducted at 0.75 V/cm for 30 min in 1 × TAE buffer supplemented with ethidium bromide. After electrophoresis, the gel was dried and assayed using Axio Imager (Zeiss) fluorescence microscope and ISIS software version 5.4.9 (MetaSystems, Altlussheim, Germany). Double-strand breaks (DSBs) were assessed by the “Tail Moment”™ (TM = tail length × DNA percentage in the tail) in the CASP software (CASP, Wroclaw, Poland).
Low melt ultra pure dna grade agarose
Manufactured by Bio-Rad
Sourced in United States
Low Melt Ultra-Pure DNA Grade Agarose is a high-quality agarose product designed for molecular biology applications. It is characterized by its low gelling temperature, making it suitable for procedures that require gentle handling of DNA samples. This agarose is rigorously purified to ensure minimal impurities, providing a reliable and consistent performance in DNA electrophoresis and other DNA-based experiments.
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Lab products found in correlation
2 protocols using low melt ultra pure dna grade agarose
1
Quantifying DNA Double-Strand Breaks in Glioblastoma Cells
2
Assessing Cellular DNA Damage Post-Chemotherapy
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Cells engrafted with ascites form of Krebs-2 tumor were injected with CP (200 mg/kg). Then, 0, 24, 30, and 42 h post CP injection, ascites cells were collected, washed in PBS and embedded in low-melting agarose plugs (Low Melt Ultra-Pure DNA Grade Agarose, Bio-Rad) in 80 µL PBS at a density of 1000 cells per block. Next, agarose blocks were immersed in a lysis buffer (50 mM EDTA, 1% N-Lauroylsarcosine [Serva], 1 mg/mL proteinase K) for 30 min at 50 °C, which was replaced with 0.5 M EDTA. Agarose-embedded cells were then washed in TE buffer (10 mM TRIS-HCl, pH 7.6; 1 mM EDTA) for 15–30 min. The blocks were similarly oriented and subjected to electrophoresis (30 min, 1 V/cm). DAPI staining was done by placing the blocks into 500 µL of DAPI solution (0.5 µg/mL), transferred onto the glass slides and analyzed under the fluorescence microscope Axiovert 40 Inverted Microscope (Zeiss).
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