STAR Protocols
Volume 2, Issue 2, 18 June 2021, 100588
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Protocol
Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis

https://doi.org/10.1016/j.xpro.2021.100588Get rights and content
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Highlights

  • A rapid protocol to generate gapped DNA molecules for single-molecule analysis

  • Customizable ssDNA gap length and position

  • Suitable for fluorescence imaging of enzymatic reactions

  • Includes protein and construct preparation, data acquisition, and analysis

Summary

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).

Subject areas

Single-molecule Assays
Microscopy
Molecular/Chemical Probes

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