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Southern Blot

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...

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Related Experiment Video

Updated: Jul 4, 2025

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

Protocol for detection of in vitro R-loop formation using dot blots.

Jack W Dowling¹, Julian R Smith², Adriana Forero¹

¹Department of Microbial Infection and Immunity, College of Medicine, The Ohio State University, Columbus, OH 43210, USA.

|January 29, 2024

Summary

This guide details a dot-blot method for detecting nucleic acids and proteins, focusing on RNA/DNA hybrids in eukaryotic cells. It outlines sample preparation and signal quantification for studying R-loop accumulation.

Keywords:

Cell culture Gene Expression Molecular Biology

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Area of Science:

Molecular Biology
Biochemistry

Background:

Dot-blot analysis offers a rapid method for detecting nucleic acids and proteins.
R-loops, structures of DNA:RNA hybrids, are implicated in various cellular processes and diseases.

Purpose of the Study:

To provide a detailed protocol for nucleic acid isolation from eukaryotic cells.
To describe sample processing for detecting RNA/DNA hybrids using dot-blot analysis.
To outline methods for quantifying dot signal intensity.

Main Methods:

Nucleic acid isolation from eukaryotic cells.
Sample preparation for dot-blotting.
Dot-blot hybridization and detection.
Quantification of dot signal intensity.

Main Results:

A comprehensive protocol for detecting RNA/DNA hybrids via dot-blot analysis is presented.
The method allows for the quantification of dot signal intensity, enabling quantitative assessment.
The protocol is adaptable for screening conditions leading to R-loop accumulation.

Conclusions:

This protocol facilitates the detection and quantification of RNA/DNA hybrids in eukaryotic cells.
The method is valuable for studying R-loop formation and its role in biological processes.
The dot-blot technique provides a convenient approach for screening and analysis.