Generation of versatile ss-dsDNA hybrid substrates for single-molecule analysis.
STAR Protoc
; 2(2): 100588, 2021 06 18.
Article
in En
| MEDLINE
| ID: mdl-34169285
ABSTRACT
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).
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
DNA
/
DNA, Single-Stranded
/
Single Molecule Imaging
Limits:
Humans
Language:
En
Journal:
STAR Protoc
Year:
2021
Document type:
Article
Affiliation country:
United kingdom
Publication country:
EEUU
/
ESTADOS UNIDOS
/
ESTADOS UNIDOS DA AMERICA
/
EUA
/
UNITED STATES
/
UNITED STATES OF AMERICA
/
US
/
USA