Northern blots
(This protocol dates from 1985 and the membranes recommended are no longer commercially available.)
1.A Hoeffer/LKB Transphor unit is recommended for Northern blots. The Bio-Rad unit has been known to melt when incorrect voltage was applied.
The recommended transfer membrane for very small RNA was 0.1 µm nylon (Schleicher & Schuell Nytran or ICN Biotrans) but this material is no longer available. RNA that is < 110 nucleotides will tend to go through a 0.2 µm membrane. Try Schleicher & Schuell nitrocellulose membranes (BA79/0.1 µm and BA75/0.05 µm)? Nylon (neutral or charged) membranes are rated for denatured > 50 bp DNA fragments, require alkaline transfer conditions and may have higher backgrounds.
Hoeffer sponges are 6” x 9” and the gel must be cut to fit these dimensions. The Hoeffer gel box can blot 4 gels simultaneously if additional cassettes are purchased.
The Hoeffer unit generates more heat than the BioRad unit.
BioRad sponges are 6 1/8” x 8 3/8” and the gel must be cut to fit these dimensions. The BioRad box is limited to a single gel. Polyacrylamide/TBE gels ranging from 4 to 8% have been successfully used, with or without urea. It is not necessary to presoak the gel to remove urea prior to blotting. If the gel is severely overloaded, some RNA will transfer to a second layer of nylon.
2.Water and buffers do not need to be DEPC-treated if they do not come from carboys. Carboy spouts tend to be a source of mildew RNases that are resistant to DEPC.
3. Prep a clean tray large enough to hold the blot cassette and soak in 0.1% DEPC or 3% hydrogen peroxide for 10 min at room temperature. Cover the pan during the soak.
4.Prep 5000 mL 1x TAE5 for the Hoeffer unit or 3300 mL 1x TAE5 for the BioRad unit. Pre-chill the buffer on ice in the coldroom.
5.Cut nylon or nitrocellulose and 2 pieces of Whatman 3MM to required dimensions while wearing gloves to prevent finger oils from coming into contact with membrane. Clip one corner to indicate orientation.
6.Drain 0.1% DEPC or 3% hydrogen peroxide from the tray. Rinse with a trace of 1x TAE5. Add remaining portion of 1 liter 1x TAE5 to tray. Add plastic gel cassette and sponges. Squeeze the air out of the sponges. Soak the membrane briefly by laying it on the sponges.
7.Open the gel plates. Apply membrane to gel and gently brush out air bubbles. Dip one piece of 3MM very briefly in 1x TAE5 and lay on top of membrane. Remove air bubbles. Lay glass plate on 3MM and flip gel over. Remove the other plate (this seems to be difficult) and prewet the remaining piece of 3MM. Lay this on top of the gel. Remove air bubbles. Move to plastic cassette and assemble holder.
8.Place the membrane side of the gel cassette towards the red electrode. Add prechilled 1x TAE5 buffer to cover. Use the 1x TAE5 buffer in the presoak tray as part of the required buffer.
