Southern Blotting to Detect Homologous Recombination in the Mouse Embryonic Stem Cell Genome

Abstract

Source: Li, J., et al. Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction. J. Vis. Exp. (2018).

In this video, we describe the Southern blotting technique to identify homologous recombination events in the genome of mouse embryonic stem cells using radiolabeled probes. DNA regions containing homologous recombination events appear as distinct bands when the membrane containing the probe-bound DNA is exposed to X-rays.

Protocol

1. Preparation of Genomic DNAs and the Digestion with Restriction Enzyme(s)

1. Prepare gDNAs from embryonic stem cells (ES cells) using a commercial kit (genomic DNA purification kit) with minor modifications.
   1. Remove media from the ES cell culture and add 500 μL of nuclei lysis solution, including RNaseA, directly to the wells to lyse the cells.
      NOTE: The cell lysate can be stored at -80 °C or treated immediately.
   2. Pipet up and down several times to lyse the cells fully and transfer them to a clean 1.5-mL tube.
   3. Add one-third of the volume of the protein precipitation solution to the 1.5 mL tu....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
QIAquick Gel Extraction Kit	QIAGEN	28704
DNA Denaturing Solution	VWR	351-013-131
DNA Neutralizing Solution	VWR	351-014-131
EcoR I	Thermo Scientific	ER0271
Dra I	Thermo Scientific	ER0221
ProbeQuan G-50 Micro Columns	GE Healthcare	28-9034-08
Hybrisol I Hybridization Solution	Millipore	S4040
Ready-To-Go DNA Labeling Beads (-dCTP)	VWR	27-9240-01
UltraPure SSC, 20x	Thermo Fisher	15557036
Salmon Sperm DNA Solution	Thermo Fisher	15632011
Whatman TurboBlotter Transfer System, Large Kits	Fisher Scientific	09-301-188
[α32P] dCTP	PerkinElmer	NEG013H100UC
Kodak X-Ray Film	Z&Z Medical	844 5702
QIAprep Spin Miniprep Kit	QIAGEN	27104
Nuclei Lysis Solution	Promega	A7941
Protein Precipitation Solution	Promega	A7951