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

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 tube, vortex vigorously for 20 s, chill the samples on ice for 5 min, and then, centrifuge at a force of 2,000 x g for 5 min. Transfer the supernatant to another clean 1.5 mL tube containing an equal volume of isopropanol; gently mix the solution. (Note that white thread-like strands can be seen at this moment.) Centrifuge at a force of 2,000 x g for 1 min; then, discard the supernatant.
   4. Wash the gDNA pellet with 1 mL of 70% ethanol at room temperature, centrifuge at a force of 2,000 x g for 1 min, aspirate the supernatant carefully, and then, air-dry the gDNA pellet for 3 min.
   5. Dissolve the gDNAs with 100 μL of DNA rehydration solution and, then, incubate at 65 °C for 1 h or at 4 °C overnight.
   6. Store the gDNAs at 2–8 °C.
2. Digest the gDNAs with predesigned RE Dra I. Set up a 30-μL digestion reaction by mixing 3 μL of 10x buffer for Dra I, 3 μL of Dra I, 10 μg of gDNAs/sample, and H₂O up to 30 μL, and incubate at 37 °C overnight.
3. Check the completeness of the digestion by DNA gel, analyzing 5 μL of the digested reaction, and then, add the 3 μL of 10x DNA-loading buffer for the subsequent step.

2. Southern Blotting

1. Southern blotting screening
   1. Separate the digested gDNAs by electrophoresis and transfer them to a membrane.
      1. Prepare a 1% agarose electrophoresis gel with ethidium bromide (EB), load the digested gDNA samples and a 1 kb ladder, and run the gel with a low voltage (30–40 V) overnight.
      2. Take out the gel and take a picture with a DNA gel-imaging system after electrophoresis. Check whether the digested and separated gDNAs display a smear-like image.
      3. Soak the gel in a tray with 0.2 N HCl solution and shake it gently for 20 min at room temperature.
      4. Transfer the gel to DNA-denaturing solution and shake it gently for 20 min at room temperature.
      5. Switch the gel into a DNA-neutralizing solution and shake it gently for 20 min at room temperature.
         NOTE: The gel is prone to breakage after this step, so it must be handled carefully.
      6. Use the rapid downward transfer system to transfer the DNAs from the gel to the membrane. Assemble the TurboBlotter and blotting stack according to the instructions provided by the manufacturer.
         NOTE: 10x or 20x saline-sodium citrate (SSC) solution is used as a transfer buffer. In general, 3 h of transfer is enough to transfer 95% of gDNAs from gel to membrane; however, a longer time of transfer is innocuous.
      7. Take out the membrane and wash it with 2x SSC for 1 min, absorb the liquid with tissues, and then cross-link the DNA with the membrane using a UV crosslinker.
         NOTE: The membrane can be stored at 4 °C for one week.
2. Label the DNA probes with radioactivity.
   1. Purify the probe plasmids using a miniprep kit according to the protocol provided by the manufacturer.
   2. Release the DNA fragments of the probes from the plasmid vector by EcoR I digestion in a reaction solution including 5 µL of buffer for EcoR I, 2 µL of EcoR I enzyme, 20 µg of plasmid DNA, and H₂O up to 50 µL, for 2 h.
   3. Run a 1% DNA gel for separating the probe DNA fragments from the vector and purify the DNA fragments of the probes with a DNA gel extraction kit according to the protocol provided by the manufacturer.
   4. Using 1 µL of the DNA solution, measure the DNA concentration of the probe DNA fragments with a spectrophotometer at a wavelength of 260/280 nm.
   5. Prepare 40-ng of probe DNAs in a 1.5 mL tube with 45 µL of TE buffer, boil for 3 min, spin briefly, and then, place the tube(s) on ice for 2 min.
   6. Add the heat-denatured probe DNAs to the tube containing ready-to-go DNA-labeling beads (-dCTP), pipet up and down to mix, add 5 µL of [α³²P]dCTP, and then, incubate at 37 °C for 15 min.
   7. Purify the labeled probes by using G-50 microcolumns according to the instructions provided by the manufacturer and, then, measure the radioactivity with a scintillation counter (optional).
3. Hybridize the membrane(s) with the labeled probes.
   1. Prehybridize the membrane.
      1. Prewarm the hybridization solution at 42 °C for 30 min. Mix 20 mL of prewarmed hybridization solution with 200 µg of boiled salmon sperm DNA in a 50-mL tube.
      2. Place the membrane into the hybridization tube. Add the mixed prehybridization solution to the hybridization tube. Place it into the hybridization oven (set rolling and the temperature at 42 °C) and let the prehybridization proceed for 30 min.
   2. Hybridize the membrane with the labeled probe(s).
      1. Take out the hybridization tube and pour the prehybridization solution into a 50 mL tube; add the denatured probe (heated at 100 °C for 3 min) from step 2.1.2.7 to this tube and mix gently.
         NOTE: Reduce any inducing bubbles.
      2. Return the mixed solution to the hybridization tube and perform the hybridization at 42 °C overnight.
4. Wash the membrane(s) to remove nonhybridized probes.
   1. Place the membrane(s) into a tray with 1x SSC + 0.1% SDS and shake gently at 55–60 °C for 10 min.
   2. Transfer the membrane(s) to a tray with 0.5x SSC + 0.1% SDS and shake gently at 55–60 °C for 10 min.
   3. Check the radioactivity on the membrane(s) by using a portable Geiger counter to decide whether a third washing is required.
5. Expose the radioactivity on the membrane to X-ray films.
   1. Remove the liquid from the washed membrane(s).
   2. Enfold the membrane(s) with plastic wrap and fix it/them in the exposure cassette.
   3. Expose the membrane to two sheets of X-ray film in a dark room.
   4. Place the exposure cassette at -80 °C overnight or longer.
6. Develop the films to visualize the results. Evaluate whether a corresponding ES clone is the desired one with the targeted recombination or not, according to the sizes of the DNA bands detected by the probes.
7. Rehybridize the same membrane with another probe after stripping off the used probe according to the following procedure: take out the used membrane, wash it 1x with clean H₂O, and then, incubate it in striping solution (55% formamide, 2% SSPE, 1% SDS, H₂O) at 65 °C with gentle shaking for 1–2 h.