These methods are based on E.
Southern. After running a gel electrophoresis, the DNA from the gel can be
transferred to a membrane via Southern Blotting.
First, stain the gel with ethidium bromide. The gel should then be treated with dilute acid to partially hydrolyze the DNA. This is to ensure that large DNA fragments are easily transferred from the gel. About 10 - 12 minute exposure to 0.25 M HCI with shaking is adequate to provide complete transfer of the DNA. The gel must not be over-treated to prevent poor quality DNA. Cover the gel with 0.5 M NaOH, 0.5 M NaCI and shake for 25 minutes in order to denature the DNA after the acid treatment. The gel can then be neutralized in 0.5 M Tris, 1.0 M NaOH, pH 7.5 for 10 minutes. Next, soak the membrane and two sheets of 3MM paper (Whatman) in 25 mM sodium phosphate, pH 6.5. Transfer the neutralized gel to a clean surface. Then apply the membrane on top of the gel, followed by the two sheets of 3MM paper and finally a stack of paper towels. Let the transfer to be for about 12 hours. Next, wash the membrane for 20 minutes in 0.1 X SSC, 0.1% SDS at 42°C with shaking. Finally, air dry on filter paper and store.
