Oneill Lab:Southern Blot

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

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In Southern analysis, DNA is transferred to a membrane and then hybridized to a probe sequence. There are many variations to how this experiment is run. This protocol is a standard experiment running digested genomic DNA on a gel prior to blotting.

Day 1

• Setup overnight digest of 11 μg DNA.

Day 2

1. Check for complete digestion with a small portion (<=1 μg) on a 0.8% agarose gel
2. Load remaining sample on a 250ml 0.8% 1x TBE agarose gel. Gel should be poured with a wide, thin comb.
3. Electrophorese overnight at 30V. If using Blue Juice loading dye, dye bands should split gel into equal thirds.
   • The blue dye band is at approximately 400bp
   • The green dye band is at approximately 6000bp

Day 3

1. Save image of gel prior to blotting
2. Prepare the wash solutions
   • Depurination
   • Denaturation
     • Must be made in a plastic container.
   • Neutralization
3. In a plastic tray, rinse gel briefly with water to remove excess buffer and ethidium bromide
4. Remove water and rinse gel with depurination solution. Rock gently for 15 minutes
5. Remove depurination solution, rinse gel with deionized water.
6. Wash gel in denaturation solution, rocking for 45 minutes.
7. Remove denaturation solution, and rinse gel with deionized water.
8. Wash gel in neutralization solution for 15 minutes
9. Remove solution and add a 2nd volume of neutralization solution. Continue to wash for 15 more minutes.
10. Remove gel to a second tray and rinse with water.
11. Prepare Southern apparatus
    1. Cut 3 pieces of Whatmann and 1 piece of Hybond-N+ equal to the size of the gel.
    2. Cut a wick of Whatmann long enough to drape over a glass plate such that the ends will be submerged, and 1/4 inch wider than the gel.
    3. Add about 1 inch of 20x SSC to a deep plastic tray.
    4. Place a clean glass plate over top of tray.
    5. Wet wick with 20x SSC and drape over glass plate. Using a glass pipet, roll out bubbles.
    6. Place gel upside down on the wick, roll out bubbles.
    7. Mark Hybond-N+ membrane and place on top of gel. Note orientation of marking to gel. Roll out any bubbles
    8. Use cling wrap to place a narrow border around the membrane. Only about 3mm should be covered on each side.
    9. Wet a piece of Whatmann with 20x SSC and place over membrane. Roll out bubbles
    10. Repeat with other two pieces of Whatmanns.
    11. Stack paper towels on top of Whatmanns.
    12. Place a tray on top of paper towels.
    13. Use a water filled falcon tube as a weight on top of the tray
    14. Leave overnight

Day 4

1. Disassemble apparatus, but leave gel and membrane attached.
2. Use an erasable pen to mark well positions on membrane.
3. Wet a piece of Whatmman larger than gel with 0.4M NaOH.
4. Place membrane on top of Whatmman, DNA side up.
5. Crosslink for 20 minutes.
6. Let membrane dry on Whatmann, DNA side up.