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 &mu;g DNA.

Day 2

 * 1) Check for complete digestion with a small portion (<=1 &mu;g) 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.
 * 4) *The blue dye band is at approximately 400bp
 * 5) *The green dye band is at approximately 6000bp

Day 3

 * 1) Save image of gel prior to blotting
 * 2) Prepare the wash solutions
 * 3) *Depurination
 * 4) *Denaturation
 * 5) **Must be made in a plastic container.
 * 6) *Neutralization
 * 7) In a plastic tray, rinse gel briefly with water to remove excess buffer and ethidium bromide
 * 8) Remove water and rinse gel with depurination solution. Rock gently for 15 minutes
 * 9) Remove depurination solution, rinse gel with deionized water.
 * 10) Wash gel in denaturation solution, rocking for 45 minutes.
 * 11) Remove denaturation solution, and rinse gel with deionized water.
 * 12) Wash gel in neutralization solution for 15 minutes
 * 13) Remove solution and add a 2nd volume of neutralization solution. Continue to wash for 15 more minutes.
 * 14) Remove gel to a second tray and rinse with water.
 * 15) Prepare Southern apparatus
 * 16) Cut 3 pieces of Whatmann and 1 piece of Hybond-N+ equal to the size of the gel.
 * 17) 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.
 * 18) Add about 1 inch of 20x SSC to a deep plastic tray.
 * 19) Place a clean glass plate over top of tray.
 * 20) Wet wick with 20x SSC and drape over glass plate. Using a glass pipet, roll out bubbles.
 * 21) Place gel upside down on the wick, roll out bubbles.
 * 22) Mark Hybond-N+ membrane and place on top of gel. Note orientation of marking to gel. Roll out any bubbles
 * 23) Use cling wrap to place a narrow border around the membrane. Only about 3mm should be covered on each side.
 * 24) Wet a piece of Whatmann with 20x SSC and place over membrane. Roll out bubbles
 * 25) Repeat with other two pieces of Whatmanns.
 * 26) Stack paper towels on top of Whatmanns.
 * 27) Place a tray on top of paper towels.
 * 28) Use a water filled falcon tube as a weight on top of the tray
 * 29) 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.