Polyacrylamide/urea gel:
40 ml per gel
0.5xTBE
6 M urea
x % polyacrylamide (19:1 acryl:bis) (the % acrylamide depends on the size of your RNA)
Mix gently (air will strongly inhibit polymerization!)
40 µl TEMED
400 µl 10% APS

Cast gel.

Add running buffer (0.5xTBE) to gel chambers. Wash out wells.
Prerun at 20W, at least 30 mins.

Heat RNA samples (in formamide LB) 95C, 1 min
Load samples.
Run gel at 20W or slower.

Set up electro-transfer:
Cut six pieces of 3MM papers and a piece of nylon membrane at 15x20cm.
Prepare 4 liters of transfer buffer: 0.5xTBE.
Transfer gel to a piece of 3MM and place on top of two pieces of 3MM under transfer buffer. Place membrane on top of gel followed by three pieces of 3MM. Remove bubbles by rolling a pipette over the gel/membrane "sandwich".
Transfer to transfer box and fill with transfer buffer (0.5xTBE).
Run at 15V, over night in cold room.

Remove membrane. Wash gently under water to remove stuck polyacrylamide.
Cross-link (RNA face up) in Stratagene crosslinker.

Probe Northern as described in the "Northern blot" protocol.

5xTBE (per liter):
54 g Tris base (450 mM)
27.5 g boric acid
1.66 g EDTA (10 mM)

JLA, 8/3/07