• Northern Blotting for Small RNAs (NWFSC)

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100x TAE buffer 5 blotting buffer for Northerns



100x TAE5 is composed of 1 M Tris base, 50 mM Na₂EDTA, 500 mM NaOAc and approximately 43 mL of glacial acetic acid (to pH 7.8) per liter of 100x buffer.

Autoclave or filter sterilize. This buffer is used with the Hoeffer/LKB apparatus to transfer RNA to nitrocellulose or nylon. The Hoeffer/LKB box requires almost 5 liters of 1x TAE₅. Gels do not need to be presoaked to remove urea. See the Northern protocol.
Acetate is oxidized to carbonate during electrophoresis, raising pH.

50x Denhardt’s

1% 5 gFicoll
1% 5 gpolyvinylpyrolidone
1% 5 gacetylated BSA
x mL Type I water
500 mL

Dissolve with mild heat and stir. Spin 2000 rpm for 15 min. Filter sterilize supernatant through 0.45 µ unit with 2 prefilters. 50x stock is difficult to filter sterilize unless prefilters are used. Store aliquots at -20℃. Do not thaw at elevated temperatures.

20x SSPE

175.3 g NaCl
27.6 g NaH₂PO₄
7.4 g Na2EDTA
800 mL Type I water
≈20.5 mL 10 N NaOH to pH 7.4
x mL Type I water
1000 mL

Autoclave or filter sterilize. SSPE can replace SSC plus NaPO₄, pH 6.7.

50% Dextran sulfate MW 5000

2 g Dextran sulfate MW 5000
x mL Type I water
4 mL

Dextran sulfate MW 5000 works just as well as the traditional MW 500,000 material, but is significantly easier to dissolve. Weigh dextran sulfate directly into anti-static treated tube (do not try to transfer from weighing paper or weigh boat).
Dextran sulfate supports the growth of bacteria. Store at -20℃

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.

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