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
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).