Protocol: RG57 genomic DNA fingerprinting
Southern Blot protocol for use with radioactive and non-radioactive hybridization
(Modified from Sambrook et al. "Molecular Cloning, 1989")
- Depurinate DNA by soaking gel in 0.25M HCl for 10 min with shaking at room temperature. Transfer the gel to a clean dish.
- Denature DNA by soaking gel in 250-500 ml of 1X Southern base solution at room temp with shaking for 15 min. Pour off solution and soak gel in another volume of 1X Southern base solution.
- Pour about 500 ml of 1X Southern base solution into a dish. Place a piece of glass or plastic across the dish to use as a support.
- Place a piece of gel blot paper across the glass support, and allow it to hang into the base solution. This will act as a wick.
- Place the gel on top of the wick, and roll it with a pipette to remove air bubbles.
- Cut a piece of nylon membrane (about the same size as the gel) and soak it in 1X Southern base solution. We use Hybond N+ membrane. Place the membrane on top of the gel.
- Cover the edge of the membrane to the edge of the dish with saran wrap on all four sides of the gel.
- Place three pieces of gel blot paper on top of the membrane. Then put a stack of paper towels on top of the gel blot paper.
- Finally, place a glass plate and a weight on top of the entire thing.
- Let the DNA transfer for 3-6 hours. Then, remove membrane from gel and place in 200 ml 1M Tris pH 7.5, 1.5M NaCl and shake for 2-3 minutes to neutralize the base solution. Membrane can be allowed to dry or can be used immediately.
0.25 M HCl
21.55 ml concentrated HCl
Add the acid to 800 ml distilled water and adjust to 1 liter.
2X Southern Base Solution
20 g NaOH
175.25 g NaCl
Add H2O to a total volume of 1 liter
