Southern Blotting to Detect Homologous Recombination in the Mouse Embryonic Stem Cell Genome
Transcription
Southern blotting involves identifying specific DNA sequences from a complex DNA mix via probe-based detection.
To identify homologous recombination or HR events in mouse embryonic stem cells with Southern blotting, obtain genomic DNA from the cells. Incubate with a restriction enzyme to digest the DNA into smaller fragments.
Load the DNA onto an agarose gel. Perform electrophoresis for size-based fragment separation.
Treat the gel with an acidic solution to remove purine bases, loosening the double-stranded DNA, dsDNA. Add alkaline denaturing solution. The high pH disrupts the hydrogen bonds, denaturing the loosened dsDNA into single-stranded DNA, ssDNA. Incubate with a neutralizing solution to neutralize the gel pH.
Assemble the transfer system using blotting papers, a nylon membrane, and gel stacked on top of the membrane. Perform the transfer. ssDNA transfers from the gel to the membrane via downward capillary action.
Irradiate the membrane with ultraviolet rays, cross-linking and immobilizing the DNA. Place the membrane in a tube containing a hybridization solution. Incubate with rotation, coating the membrane and preventing nonspecific probe binding.
Finally, incubate the membrane with the hybridization buffer containing single-stranded radioactively-labeled probes. The probes bind to specific DNA regions via sequence complementarity.
Wash the membrane, removing unbound probes. Expose the membrane to X-rays. Distinct bands on the membrane distinguish DNA regions containing HR events from control regions.
