DNA fiber analysis was performed as described (14 (link), 26 (link)). 5-Iodo-2′-deoxyuridine (IdU; 50 μM; Sigma-Aldrich, I7125) was used to label the cells for 30 min, with or without HU (Selleck, S1896) treatment, and then 250 μM 5-chloro-2′-deoxyuridine (CldU; Sigma-Aldrich, C6891) was used for the second labeling. After labeling, ~3000 cells in 2.5 μl of suspension were dropped onto one end of the glass slide, mixed with 7.5 μl of lysis buffer [50 mM EDTA, 0.5% SDS, and 200 mM tris-HCl (pH 7.5)], and incubated for 8 min at room temperature. Then, the slides were tilted to 15° to spread the DNA fibers along the slide. The slides were then treated with 2.5 M hydrochloric acid, incubated with rat anti-BrdU/CIdU (BU1/75) monoclonal antibody (Novus, NB500-169), and mouse anti-IdU monoclonal antibody (BD, 347580). The secondary antibodies were Alexa Fluor Cy3–conjugated goat anti-rat and Alexa Fluor 488–conjugated goat anti-mouse, respectively. Images were captured using a confocal microscope (Olympus, FV1000). ImageJ software was used to measure the length of DNA fibers, and the formula that 1 μm = 2.59 kb was used to convert the micrometer value to kilobase. New replication origin firing analysis was performed as described (54 ). Cells were labeled with CIdU for 10 min (without HU) or with 100 μM HU for 20 min.
Nb500 169
Manufactured by BD
The NB500-169 is a laboratory equipment product that serves as a centrifuge. It is designed to separate different components within a liquid sample through the application of centrifugal force. The NB500-169 operates at a maximum speed of 16,000 revolutions per minute and can accommodate a variety of sample sizes and types.
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3 protocols using nb500 169
1
Quantifying DNA Replication Dynamics
2
Dual-Labeling Assay for Replication Dynamics
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Cells were labeled with 50 μM 5-iodo-2′ -deoxyuridine (IdU; Sigma, I7125) for 30 min and then incubated with 50 μM 5-chloro-2′ -deoxyuridine (CldU; Sigma, C6891) for 30 min, with or without treatment of hydroxyurea (HU, Selleck, S1896). DNA fibers were made as described7 (link). Briefly, cells were harvested and suspended in PBS at a concentration of 106/mL. 2,500 cells were lyzed in 12 μL of spreading buffer (0.5% SDS, 50 mM EDTA, 200 mM Tris, pH 7.4) on one end of the glass slide. DNA fibers were spread along the slide by tilting the slides, fixed with freshly prepared methanol-acetic acid (3:1), and treated with 2.5 M hydrochloric acid. For detection of labeled fibers, rat anti-BrdU/CIdU (BU1/75) monoclonal antibody (Novus, NB500-169) and mouse anti-IdU monoclonal antibody (BD, 347580) were used as the primary antibody. The secondary antibodies were AlexaFluor 488-conjugated goat anti-mouse IgG or Cy3-conjugated goat anti-rat IgG. Images were captured from Olympus confocal microscope. The length of labeled fibers were measured using the Image J software, and values were transformed into kilobases using the transform factor 1 μm = 2.59 kb. At least 200 fibers were analyzed for stalled fork restart and nascent DNA degradation.
3
Immunofluorescent Staining and DNA Fiber Analysis
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