Southern Blot

1. Run gel (0.8 -1.0% agarose is best). For yeast chromosomal Southerns, digest 20 μg DNA. Use 50 ml minigel for most purposes. Photograph gel, but minimize exposure to UV light.2. Depurination: Place gel in 250 mls 0.25M HCl.(250 mls = 11 mls 5.8M HCl + 250 mls ddH2O)Agitate gently on rotator for 10 minutes after dye front turns yellow.3. Decant HCl. Wash briefly with ddH2O.4. Denaturation: Place gel in 250 mls 1.5M NaCl + 0.5M NaOH.(500 mls NaCl/NaOH = 44 g NaCl + 10 g NaOH)Rotate gently 20 minutes.Decant, add 250 mls fresh NaCl/NaOH, rotate 20 minutes.5. Decant NaCl/NaOH. Wash briefly with ddH2O.6. Neutralization: Place gel in 250 mls of 3M NaCl + 0.5M Tris, pH 7.0.500 mls = 88 g NaCl + 3 g Tris base + 36 g Tris HClRotate gently 20 minutes.Decant, add 250 mls fresh NaCl/Tris, rotate 20 minutes.7. Measure gel exactly. Cut off bottom right corner. Put gel back in neutralization solution. Cut nitrocellulose paper about 1 mm shorter than gel and wet in ddH2O. Soak nitrocellulose paper 5-10 minutes in 20X SSC. Cut 2 sheets of THIN filter paper 1 mm shorter than nitrocellulose paper.Cut 2 sheets of THICK paper 1 mm shorter than nitrocellulose. Cut 2-3 inch stack of paper towels.8. Set up Transfer. Use 2 pieces of thin filter paper to make wick. Place wick over glass plate on support platform. Remove air bubbles at all steps in the set up procedure. Put gel face down on wick. Put wetted nitrocellulose paper down correctly first time. Put on thin paper pieces wetted in 20X SSC. Assemble transfer as depicted below. Use 50-100 g weight on top. Glass plates work well. Place plastic wrap around gel and wick to prevent evaporation.9. After transfer, wash filter 5 minutes in 4X SSC. Remove excess liquid. Air dry for 30 minutes.Bake at 70°C in vacuum oven for 2 hours. Blot can be stored under vacuum wrapped in foil.