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Northern Blot: Gel Transfer

Adapted from Molecular Cloning (Maniatis) and Current Protocols in Molecular Biology.


Objective:

Northern blotting allows detection of specific RNA sequences. RNA is fractionated by agarose gel electorphoresis, followed by transfer (blotting) to a membrane support, followed by hybridization with DNA or RNA probes.

Procedures:

Start=> Run RNA gel either formaldehyde or glyoxal
 

  1. If the gel is formaldehyde, rinse 3x10 minutes in RNase-free H2O.
  2. Soak gel in transfer buffer (20X SSC for nitrocellulose membranes and 10X SSC for nylon membranes) for 2X15 minutes.
  3. Set up transfer.
    1. Place solid support with Whatman 3MM filter paper wicks in a pool of transfer buffer. Optionally, firm clean sponges covered with a layer of Whatman 3MM can replace the solid support and wick typically used.
    2. Wet Whatman 3MM with transfer buffer.
    3. Place four strips of plastic wrap on Whatman 3MM paper so that the exposed filter paper is just smaller than the gel. Alternatively, a square just smaller than the gel can be cut from a sheet of plastic wrap. This prevents "short circuiting" of the buffer around the gel.
    4. Center gel above solid support or sponges and lower onto Whatman 3MM paper, remove any air bubbles.
    5. Cut the appropriate membrane to the size of the gel. For a nylon membrane wet and allow to soak in water for 5 minutes; for nitrucellulose, soak for ten minutes with 20X SSC. Place on the gel avoiding air bubbles. Do not handle membrane with hands- gloved or not. Use a clean blunt-ended forceps instead
    6. Flood the membrane with transfer buffer and cover with 5 sheets of Whatman 3MM filter paper cut to the same size as the membrane.
    7. Cut paper towels to the same size as the membrane and Whatman 3MM above the gel, and stack to a height of 4cm above the gel.
    8. Cover with a glass plate, and add a weight to hold everything in place (not too much weight it could crush the gel).
  4. Allow transfer to go overnight.
  5. Remove paper towels and filter papers. Recover the membrane and flattened gel. Mark in pencil the position of the wells and orientation of the membrane (an asymmetric cut at the corner is sufficient).
  6. For nylon membranes, cover in UV-transparent plastic wrap, place RNA-side-down on a UV transilluminator (254nm), and UV irradiate to cross-link.
    For nitrocellulose membranes, place membrane between two sheets of Whatman 3MM filter paper and bake in a vacuum oven for 2 Hr at 80 degrees Celsius.
  7. To stain transferred RNA (a ladder, for instance). Nylon membranes can be soaked in 0.03% methylene blue, 3M sodium acetate, pH 5.2 for 45 seconds. Followed by destaining in water for 2 minutes.
  8. Store membranes dry between sheets of Whatman 3MM filter paper. Long term storage should be in a desiccator at room temperature or at 4 degrees Celsius.