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Merck

A laser-mediated photo-manipulative toolbox for generation and real-time monitoring of DNA lesions.

STAR protocols (2021-08-18)
Bindhu K Madhavan, Zhe Han, Albert Sickmann, Rainer Pepperkok, Peter P Nawroth, Varun Kumar
RESUMEN

With the advancement of laser-based microscopy tools, it is now possible to explore mechano-kinetic processes occurring inside the cell. Here, we describe the advanced protocol for studying the DNA repair kinetics in real time using the laser to induce the DNA damage. This protocol can be used for inducing, testing, and studying the repair mechanisms associated with DNA double-strand breaks, interstrand cross-link repair, and single-strand break repair. For complete details on the use and execution of this protocol, please refer to Kumar et al. (2017, 2020).

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Disolución salina tamponada con fosfatos de Dulbecco, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
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Seroalbúmina bovina, heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98%
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L-Glutamina solution, 200 mM, solution, sterile-filtered, BioXtra, suitable for cell culture
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Penicilina- Estreptomicina, with 10,000 units penicillin and 10 mg streptomycin per mL in 0.9% NaCl, 0.1 μm filtered, BioReagent, suitable for cell culture
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5-Bromo-2′-desoxiuridina, BioUltra, ≥99%
Millipore
Triton X-100, Non-ionic detergent and emulsifier. Has an optimal pH range of 6.0-8.0.
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bisBenzimide H 33342 trihydrochloride, ≥98% (HPLC and TLC)
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Medio de Eagle modificado de Dulbecco, poca glucosa, With 1000 mg/L glucose, L-glutamine and sodium bicarbonate, without phenol red, liquid, sterile-filtered, suitable for cell culture