1. Treat gel with 250 ml 0.25M HCl (shaking). The length of acid treatment varies depending on the thickness of the gel. For a 3mm gel 5 min is good, for an 8 mm gel try 15 min.

2. Repeat incubation in acid.

4. Denature and cleave depurinated DNA by incubating gel in 0.5M NaOH/1.0M NaCl (250 ml) 15 min. with shaking. Repeat.

6. Neutralize 30 min in 0.5M Tris-HCl, pH 7.4/3M NaCl with shaking. Repeat.

7. Transfer gel to a tray of 10X SSC. This is your transfer buffer.

8. Cut nitrocellulose to the exact size of the gel. Float the cut nitrocellulose on water being careful to avoid trapping air bubbles between the water and the nitrocellulose. It is important to always wear gloves when handling nitrocellulose to avoid getting oils from your skin onto the membrane. Then carefully submerge the membrane in 10 X SSC. Nitrocellulose will not wet if you put it directly into high salt such as 10 X SSC. The high salt buffer facilitates binding of the DNA to the filter. Go to "setting up the blot".

1. Prepare

1.0 l of 0.25 M HCl

1.0 l of 0.4 M NaOH (this is your transfer buffer)

2. Soak gel in 2 volumes 0.25 M HCl with gentle agitation until the bromophenol blue tracking dye turns yellow (usually 10-15 min.), then rinse briefly in distilled water.

3. Cut genescreen membrane to the dimensions of the gel, mark the orientation of the membrane with a soft pencil- the concave side when the membrane is dry (convex side after the membrane is wetted) is the side which goes against the gel. Carefully wet the genescreen in distilled water. Go to "setting up the blot".

1. Cut a sheet of Whatman 3MM filter paper to the width of the gel, but 5 inches longer than the length of the gel. This is your wick.

Cut 5 sheets of Whatman 3MM filter paper to the exact size of the gel.

Cut a stack of paper towels (about 6 cm high) in the dimensions of the gel

2. Fill a tray that easily fits your gel with transfer buffer. In the tray, place a solid support (such as an inverted plastic box) with the wick saturated in transfer buffer on top. Roll out any bubbles between the box and the wick with a test tube. Alternatively, place in the tray 4 non-cellulose sponges saturated in transfer buffer (see diagram).

3. Prewet a 3MM filter in transfer buffer and place on the wick or on the sponges. Remove bubbles by repeatedly rolling a test tube over the filter.

4. Saturate the filter with transfer buffer, then carefully place the gel onto the pad, taking care that no bubbles are trapped beneath the gel.

5. Pour transfer buffer onto the surface of the gel, filling the sample slots and flooding the gel surface (images of the sample slots are retained in transfer and hybridization, allowing unequivocal identification of the origin).

6. Cover all exposed 3MM paper with Saran wrap or parafilm. This prevents any short circuiting of transfer buffer passing around the gel. All of the passage of transfer buffer should be through the gel.

7. Lower the sheet of prewetted nitrocellulose or genescreen onto the gel surface. Contact first in the center, then allow the edges to gradually fold down and make sure there are no bubbles between the gel and the nitrocellulose or genescreen.

8. Flood the surface with transfer buffer. Place two prewetted 3MM filters on top of the nitrocellulose or genescreen. Remove bubbles. Place the remaining dry sheets of 3MM paper onto the stack, followed by the paper towels. Place a glass plate or heavy book on the top of the stack.

9. Transfer for 2 hours to overnight, depending on how much DNA is in the gel. After transfer, air dry then bake nitrocellulose for 1 hour at 80 degrees in the vaccuum oven. Genescreen does not need to be baked.

Notes:

(I) This method can also be used for transferring RNA from a formaldehyde gel. Shake gel in 10X SSC for 20 minutes after running. Do not stain. Blot immediately as above, staring at "setting up the blot".

20 X SSC:

3M NaCl

0.3M Na Citrate, pH 7.0