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

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.

For each gDNA, mix 30 microliters of 10X buffer for Dra I, three microliters of Dra I, and 10 micrograms of gDNAs per sample. Add water until the final volume is 30 microliters, and incubate at 37 degrees Celsius overnight to digest the gDNAs.

Prepare a 1% agarose electrophoresis gel with ethidium bromide. Load the previously-acquired samples along with a 1 Kb ladder onto the gel, and run it at 30 to 40 volts overnight. The next day, soak the gel in a tray, containing a 0.2 Newton hydrochloric acid solution. Use a shaker to shake the tray gently for 20 minutes at room temperature.

Transfer the gel to a tray containing a DNA-denaturing solution. Shake it gently for 20 minutes at room temperature. Then, transfer the gel into a tray containing a DNA-neutralizing solution, and shake it gently for 20 minutes at room temperature. Using a rapid downward transfer system, transfer the DNAs from the gel to the membrane.

Next, assemble the TurboBlotter and blotting stack as outlined in the instructions provided by the manufacturer. After this, take out the membrane and wash it with 2X saline-sodium citrate for one minute. Absorb the liquid with tissues, and then, use a UV cross-linker to crosslink the DNA with the membrane.

To begin labeling the DNA probes with radioactivity, add the heat-denatured probe DNAs to the tube containing the ready-to-go DNA-labeling beads. Pipette up and down to mix, and add 5 microliters of radioactively-labeled deoxycytosine triphosphate. Incubate at 37 degrees Celsius for fifteen minutes. Purify the labeled probes by using G-50 microcolumns, according to the instructions provided by the manufacturer. Use a scintillation counter to measure the radioactivity.

To begin hybridizing the membranes with the labeled probes, place the membrane into the hybridization tube. Add the mixed pre-hybridization solution. Place the tube into hybridization oven, and let the pre-hybridization proceed at 42 degrees Celsius for 30 minutes.

Then, remove the tube from the oven, and pour the pre-hybridization solution into a 50-milliliter tube. Add the denatured probe and mix gently. Add the mixed hybridization solution into the hybridization tube, and return the tube into the hybridization oven. Then, let the hybridization proceed overnight.

To remove the non-hybridized probes, place the membrane into a tray containing 1x saline-sodium citrate with 0.1% SDS and shake gently at 55 to 60 degrees Celsius for 10 minutes. After this wrap the membrane with plastic wrap, and fix it in the exposure cassette. In a dark room, expose the membrane to 2 sheets of X-ray film. Develop the film to visualize the results.