Southern Blotting

1. Transfer the DNA in the gel onto a positively charged nylon membrane (Hybound +) by Southern blotting.

2. First the gel is pretreated by soaking in a series of solutions to denature and neutralize the DNA and gel matrix:

a. Place gel in a clean dish containing: 200 mL 0.5 M NaOH/1.5 M NaCl (denature).

b. Shake slowly on a platform shaker for 20 min at room temperature.

c. Replace with fresh denaturation buffer and shake for an additional 20 min.

d. Pour off buffer and add: 200 mL 0.3 M Tris/3 M NaCl, pH 7.0 (neutralize).

e. Shake as before for 20 min, replace with fresh neutralization buffer and shake another 20 min.

f. The gel is then blotted overnight onto a membrane in neutralization buffer.

3. After overnight blotting remove the membrane from the gelstack.

4. Rinse the membrane briefly with 2X SSC, place it on a sheet of dry Whatman 3 MM paper and allow it to dry.

5. To immobilize the DNA, it is cross linked onto the membrane by UV in a UV cross-linker (use setting suggested by the manufacturer).

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