A transcription-based approach to purify R-loop-containing plasmid DNA templates in vitro

STAR Protoc. 2023 Mar 17;4(1):101937. doi: 10.1016/j.xpro.2022.101937. Epub 2022 Dec 13.

Abstract

To study the direct effects of R-loops on DNA replication and other DNA-templated processes in vitro, R-loop-containing DNA templates need to be prepared efficiently and to near homogeneity. Here, we describe a simple transcription-based approach to form R-loops on plasmid DNA templates in vitro. We detail steps to transcribe a DNA sequence element with a high propensity to form co-transcriptional R-loops using T7 RNA polymerase. We describe nucleolytic digestion of free RNA, deproteinization, and repurification of R-loop-containing templates via gel filtration. For complete details on the use and execution of this protocol, please refer to Kumar et al.1.

Keywords: Molecular Biology; Protein Biochemistry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • DNA / genetics
  • Plasmids / genetics
  • R-Loop Structures*
  • RNA
  • Transcription, Genetic*

Substances

  • RNA
  • DNA