Assembling the blotting apparatus
Fixing the DNA to the blot
- After the transfer is complete, remove the weight, paper towels, and the two sheets of filter paper. Turn over the gel and the blotting membrane together, and lay them, gel-side up, on a sheet of dry filter paper. Mark the positions of the gel lanes on the membrane using a ballpoint pen or a soft-lead pencil. Peel the gel from the membrane. If desired, keep the gel to assess the efficiency of DNA transfer, otherwise discard. Before removing the gel from the blotting membrane, ensure that the gel lanes are marked so that they can be later identified. In order to assess the efficiency of DNA transfer, stain the gel with ethidium bromide after blotting to see how much DNA remains.
- Fix the DNA to the blot, either by baking (see step 3) or by UV-crosslinking
(see step 4).
Tip: UV-crosslinking generally gives better results and enhanced sensitivity compared with baking. However, effective crosslinking requires optimization of the system.
- Use either this step or step 4: To fix the DNA to the membrane by baking, first let the blot air-dry on a sheet of filter paper, then place between two sheets of filter paper, and bake at 80°C for 2 h. Proceed to step 5.
- Use either this step or step 3: To fix the DNA by UV-crosslinking, first protect the surface of the membrane by covering the UV source (e.g., a transilluminator) with plastic wrap. Then take the damp blot and expose the side with DNA to the UV source for a predetermined length of time. Proceed to step 5.
Tip: It is important to optimize the system for UV-crosslinking. To do this, prepare a blot with several control DNA samples. Cut the blot into separate strips for each lane, and irradiate each blot for different times, varying from 0.5 to 5 min. Hybridize all the blots together and determine which time gives the optimal signal intensity. It is important to use the same conditions (UV wavelength, distance from UV source) for each experiment. It is also important to calibrate the system routinely, as the energy emitted from a UV bulb is reduced with use.
Tip: UV light can damage the eyes and skin. Always wear suitable eye and face protection.
- If the blot will not be used immediately, store it at room temperature covered in plastic wrap.