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

Published: May 31, 2023

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) Prepare gDNAs from embryonic stem cells (ES cells) using a commercial kit (genomic DNA purification kit) with minor modifications. 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. Pipet up and down several times to lyse t…

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
Southern Blotting to Detect Homologous Recombination in the Mouse Embryonic Stem Cell Genome. J. Vis. Exp. (Pending Publication), e21370, doi: (2023).

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