The procedure is specifically designed for blotting an agarose gel onto an uncharged or positively charged nylon membrane. With the minor modifications detailed in the anno tations, the same protocol can also be used with nitrocellulose membranes.

The protocol is divided into three stages. First, the agarose gel is pretreated by soaking in a series of solutions that depurinate, denature, and neutralize the DNA and gel matrix. The second stage is the transfer itself, which occurs by upward capillary action. Finally, the DNA is immobilized on the membrane by UV irradiation (for nylon) or baking (for nitrocellulose).

Materials

DNA samples to be analyzed	0.25 M HCl
Denaturation solution: 1.5 M NaCl/0.5 M NaOH (store at room temperature)	Neutralization solution: 1.5 M NaCl/0.5 M Tris·Cl, pH 7.0 (store at room temperature)
20× and 2× SSC (APPENDIX 2)	Oblong sponge slightly larger than the gel being blotted
Whatman 3MM filter paper sheets	Nylon or nitrocellulose membrane
UV transilluminator or UV light box for nylon membranes	Additional reagents and equipment for restriction endonuclease digestion and agarose gel electrophoresis

Prepare the gel

1. Digest the DNA samples with appropriate restriction enzyme(s), run in an agarose gel with appropriate DNA size markers, stain with ethidium bromide, and photograph with a ruler laid alongside the gel so that band positions can later be identified on the membrane.

2. Rinse the gel in distilled water and place in a clean glass dish containing ∼10 gel volumes of 0. 25 M HCl. Shake slowly on a platform shaker for 30 min at room temperature.

3. Pour off the HCl and rinse the gel with distilled water. Add ∼10 vol denaturation solution and shake as before for 20 min. Replace with fresh denaturation solution and shake for a further 20 min.

4. Pour off the denaturation solution and rinse the gel with distilled water. Add ∼10 vol neutralization solution, shake as before for 20 min, then replace with fresh neutrali zation solution and shake for a further 20 min.

Set up the transfer

5. place an oblong sponge, slightly larger than the gel, in a glass or plastic dish (if necessary, use two or more sponges placed side by side). Fill the dish with enough 20× SSC to leave the soaked sponge about half-submerged in buffer.

6. Cut three pieces of Whatman 3MM paper to the same size as the sponge. Place these on the sponge and wet them with 20× SSC.

7. Place the gel on the filter paper and squeeze out air bubbles by rolling a glass pipet over the surface.

8. Cut four strips of plastic wrap and place over the edges of the gel.

9. Cut a piece of nylon membrane just large enough to cover the exposed surface of the gel. Pour distilled water ∼0. 5 cm deep in a glass dish and wet the membrane by placing it on the surface of the water. Allow the membrane to submerge, then leave for 5 min.

10. Place the wetted membrane on the surface of the gel. Try to avoid getting air bubbles under the membrane; remove any that appear by carefully rolling a glass pipet over the surface.

11. Flood the surface of the membrane with 20× SSC. Cut five sheets of Whatman 3MM paper to the same size as the membrane and place these on top of the membrane.

12. Cut paper towels to the same size as the membrane and stack these on top of the Whatman 3MM papers to a height of ~4 cm.

13. Lay a glass plate on top of the structure and place a weight on top to hold everything in place. Leave overnight.

Disassemble the transfer pyramid

14. Remove the paper towels and filter papers and recover the membrane. Mark in pencil the position of the wells on the membrane and ensure that the up-down and back-front orientations are recognizable.

15. Rinse the membrane in 2× SSC, then place it on a sheet of Whatman 3MM paper and allow to dry.

16. Wrap the membrane UV-transparent plastic wrap, place DNA-side-down on a UV transilluminator (254-nm wavelength) and irradiate for the time determined from the support protocol.

17. Store membranes dry between sheets of Whatman 3MM paper for several months at room temperature. For long-term storage, place membranes in a desiccator at room temperature or 4°C.