How to carry out Northern blotting
Following electrophoresis, the formaldehyde/agarose gel is placed in water for 15 minutes with light shaking. This is followed by two 15-minute rinses in 10xSSC. The next stage in Northern blotting involves the gel being placed upon two pieces of pre-wetted Whatmann 3MM paper, supported over a reservoir of 10xSS C. The ends of the Whatmann 3MM paper are submerged in the 10xSSC solution to form a wick. A piece of nylon membrane (Hybond-NX, Amersham) should be cut to the same size as the gel, and immersed in 10xSSC for 15 minutes. It is then placed on top of the gel, and any air-bubbles present removed by rolling with a glass pipette.
Three further pieces of Whatmann 3MM paper are cut to the size of the gel, pre-soaked in 10xSSC and placed on top of the nylon membrane. A stack of paper towels are then placed on top, and a ~500g weight was placed on the towels. This encourages the transfer of the 10xSSC from the reservoir, to the paper towels, though a capillary action. The gel should be left overnight, to allow the RNA to thoroughly transfer to the membrane. After the northern blotting transfer, RNA is bound to the membrane by UV-crosslinking (1200 m J UV light, Stratagene Stratalinker), or by baking for 2 Hrs at 80°C.