Oneill Lab:Southern Blot
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Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology
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.
- Setup overnight digest of 11 μg DNA.
- Check for complete digestion with a small portion (<=1 μg) on a 0.8% agarose gel
- Load remaining sample on a 250ml 0.8% 1x TBE agarose gel. Gel should be poured with a wide, thin comb.
- 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
- Save image of gel prior to blotting
- Prepare the wash solutions
- Must be made in a plastic container.
- In a plastic tray, rinse gel briefly with water to remove excess buffer and ethidium bromide
- Remove water and rinse gel with depurination solution. Rock gently for 15 minutes
- Remove depurination solution, rinse gel with deionized water.
- Wash gel in denaturation solution, rocking for 45 minutes.
- Remove denaturation solution, and rinse gel with deionized water.
- Wash gel in neutralization solution for 15 minutes
- Remove solution and add a 2nd volume of neutralization solution. Continue to wash for 15 more minutes.
- Remove gel to a second tray and rinse with water.
- Prepare Southern apparatus
- Cut 3 pieces of Whatmann and 1 piece of Hybond-N+ equal to the size of the gel.
- 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.
- Add about 1 inch of 20x SSC to a deep plastic tray.
- Place a clean glass plate over top of tray.
- Wet wick with 20x SSC and drape over glass plate. Using a glass pipet, roll out bubbles.
- Place gel upside down on the wick, roll out bubbles.
- Mark Hybond-N+ membrane and place on top of gel. Note orientation of marking to gel. Roll out any bubbles
- Use cling wrap to place a narrow border around the membrane. Only about 3mm should be covered on each side.
- Wet a piece of Whatmann with 20x SSC and place over membrane. Roll out bubbles
- Repeat with other two pieces of Whatmanns.
- Stack paper towels on top of Whatmanns.
- Place a tray on top of paper towels.
- Use a water filled falcon tube as a weight on top of the tray
- Leave overnight
- Disassemble apparatus, but leave gel and membrane attached.
- Use an erasable pen to mark well positions on membrane.
- Wet a piece of Whatmman larger than gel with 0.4M NaOH.
- Place membrane on top of Whatmman, DNA side up.
- Crosslink for 20 minutes.
- Let membrane dry on Whatmann, DNA side up.