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Protocol: RG57 genomic DNA fingerprinting

Southern Blot protocol for use with radioactive and non-radioactive hybridization

(Modified from Sambrook et al. "Molecular Cloning, 1989")

  1. Depurinate DNA by soaking gel in 0.25M HCl for 10 min with shaking at room temperature. Transfer the gel to a clean dish.
  1. 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.
  1. 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.
  1. 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.
  1. Place the gel on top of the wick, and roll it with a pipette to remove air bubbles.
  1. 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.
  1. Cover the edge of the membrane to the edge of the dish with saran wrap on all four sides of the gel.
  1. 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.
  1. Finally, place a glass plate and a weight on top of the entire thing.
  1. 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

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