By Zhou Songyang (auth.), Zhou Songyang (eds.)
New and quick advances in know-how have outfitted us with a number of instruments and systems to invite basic questions of telomere legislation and feature allowed investigators to hold out experiments utilizing different version platforms. for instance, proteomic, genomic, and molecular ways have afforded us remarkable perception into the advanced protein interplay networks at paintings at the telomere chromatin and the targeted information about telomere dynamics in line with rigidity or stimuli. Telomeres and Telomerase: tools and Protocols, moment Edition builds upon the telomerase assays featured within the well known first version to surround many various assays that permit investigators to question the functionality of telomere proteins and the responses of the telomere DNA, together with special examinations of biochemical, molecular, and proteomic techniques. Written within the hugely winning Methods in Molecular Biology™ sequence layout, chapters comprise introductions to their respective themes, lists of the required fabrics and reagents, step by step, conveniently reproducible laboratory protocols, and professional tips about troubleshooting and heading off identified pitfalls.
Authoritative and sensible, Telomeres and Telomerase: equipment and Protocols, moment Edition serves as an incredible, up to date advisor for investigators extra pursing this very important box of study.
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Additional resources for Telomeres and Telomerase: Methods and Protocols
Recover the cell suspension in PBS. 2 mL above the cell pellet. Resuspend the cell pellet in the remaining supernatant. Hypotonic shock: Slowly add (drop-wise) the pre-warmed hypotonic solution to the resuspended cells while gently vortexing. Incubate at 37°C for 30–40 min (see Note 11). Prefixation: Add a few drops of freshly prepared fixative ethanol/acetic acid (3/1, V/V) and invert to mix. Spin the cells down at slow speed (200 rcf, 5 min). Remove most of the supernatant and resuspend the cell pellet in the remaining liquid.
While both the G and C probes can be used, the G probe generally yields better and stronger signals. 5–1 × 106 cpm/mL. The membrane is first washed in low-stringency buffer once at room temperature and once at 37°C, and then in highstringency buffer at least twice at room temperature. The stringency of the wash may be further raised by increasing the number of washes, or the temperature for high-stringency wash (to 37°C or 50°C if needed). The membrane should be checked with a Geiger counter periodically.
Wash three times 5 min in PBS 1×. 5 mg/mL in PBS) for 15 min. Quick wash in PBS 1×. Expose slides to a 306 nm UV source for 30 min. Typically, the slides are placed on a heating plate (55°C) covered with excess PBS and a coverslip (to avoid desiccation), and placed under a flipped-over UV box. The slides should be 1 cm away from the UV source. After UV exposure, immerse slides in PBS 1× to help the coverslip to come off and the slides to cool down. After draining all the PBS, treat slides with 3 U/mL of ExoIII for 5 min at room temperature (use at least 40 mL/slide and put a coverslip on to ensure even distribution) (see Note 17).