Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis
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Ondrej Belan George Moore Artur Kaczmarczyk Matt Newton Roopesh Anand Simon Boulton David S RuedaAbstract
Here, we describe a rapid and versatile protocol to generate gapped DNA substrates for single-molecule (SM) analysis using optical tweezers via site-specific Cas9 nicking and force-induced melting. We provide examples of single-stranded (ss) DNA gaps of different length and position. We outline protocols to visualize these substrates by replication protein A-enhanced Green Fluorescent Protein (RPA-eGFP) and SYTOX Orange staining using commercially available optical tweezers (C-TRAP). Finally, we demonstrate the utility of these substrates for SM analysis of bidirectional growth of RAD-51-ssDNA filaments. For complete details on the use and execution of this protocol, please refer to Belan et al. (2021).
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Journal STAR Protocols
Volume 2
Issue number 2
Pages 100588
Available online
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Publisher website (DOI) 10.1016/j.xpro.2021.100588
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Europe PubMed Central 34169285
Pubmed 34169285
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