To transfer DNA from a gel slab to a filter for subsequent testing.
A restriction analysis (A-28) is usually performed prior to the transfer.
In the movie, you will see an experienced laboratory worker handling gels. Begin to learn this technique yourself by casting 2 or 3 gels and practicing the manipulation of those gels through the procedures shown.
Wear gloves. Use a fresh razor blade. Trim the gel to remove wells. Cut a corner to permit orientation with respect to the gel. (Gloves are worn to prevent contamination of the gel.) Measure the gel.
Cover the gel with 0.25 M hydrochloric acid. (This acid treatment "nicks" the DNA and thereby facilitates the transfer of large fragments. The duration controls the extent of "nicking." Treatment for too long causes the fragments to be too small.) Close the tray cover. Shake gently for about 8 minutes. Drain the hydrochloric acid into the sink. Repeat the acid treatment procedure, with shaking. The gel is washed with distilled water to remove acid. Shake with distilled water. Drain the wash water into the sink.
Add 0.5 M NaOH/1.0 M NaCl. Shake for 15 minutes. Drain. Repeat one time. Wash with distilled water. This step denatures the DNA so that it will "stick" to nitrocellulose paper. Wash the gel for 30 minutes with a solution of Tris buffer and sodium chloride. Repeat this treatment. Wash briefly with distilled water.
The nitrocellulose is supplied as sheets sandwiched between two sheets of protective paper. It is extremely important not to touch the nitrocellulose with bare fingers. Grease will prevent the paper from wetting properly. Using gloved hands, a ruler, pencil, and scissors (or razor or paper cutter), measure and cut out a piece of nitrocellulose identical in size to the trimmed gel. Treat the nitrocellulose with a solution of 20x SSC.
As a template, use a sponge cut slightly larger than the gel. Cut two pieces of paper (e.g., Whatman 3MM). Place the 20x SSC solution in a tray. Soak the sponge in that solution. Place one precut paper sheet over the soaked sponge. The paper will absorb the solution. Squeeze out air bubbles by stroking. A pipet may be used to "roll down" the nitrocellulose without trapping air bubbles. Air bubbles between the sponge and the papers above the nitrocellulose will prevent transfer of the DNA. Place a second sheet of paper over the first.
Use a plastic spatula to transfer the treated gel to the paper. If air bubbles are trapped, remove the gel and start again. Cover the entire system with clear plastic wrap. Cut a slot exposing the entire gel surface to the air. Adjust the plastic wrap by tamping to the surface. (Saran Wrap® works satisfactorily.) Pick up the nitrocellulose paper with forceps. Cut off a corner. Orient the nitrocellulose on top of the gel. Take care that no air bubbles are trapped, and that there is contact between the nitrocellulose and the gel.
Place the two pieces of paper cut to gel size on top of the nitrocellulose. Place a stack of paper towels precut to the gel size on top. Place a weight on top of the stack. Check for stability after 15 minutes, and again after 1 hour. Adjust if necessary.
Leave overnight. Remove the paper towels and the two paper sheets. Use forceps to place the nitrocellulose in a 2x SSC solution. Wait one hour. Set the gel aside on paper for drying. Discard gel.
Remove the nitrocellulose and place onto a folded sheet of Whatman 3MM paper to air dry. Wait for the nitrocellulose to air dry, about 1-1.5 hours. Place the dried nitrocellulose in a vacuum oven to bake at 80°C and 25 kPa for 2 hours. Transfer the baked nitrocellulose to a plastic freezer bag. Seal with a thermal heating device used to seal sandwich bags.
The nitrocellulose is saved under refrigeration for hybridization (A-26) experiments.