Running a Urea Gel for Northern Blot Analysis

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Running a Urea Gel for Northern Blot Analysis

1. Pour a 6% polyacrylamide gel similar to pouring one for SDS Electrophoresis, except substituting SDS with 8.3 M urea, not having a stacking gel, and using TBE buffer instead of Tris and Glycine. The urea can be initially dissolved in the correct amount of TBE (45 mM Tris, pH 8, 45 mM boric acid, 1 mM EDTA) buffer via heating in a microwave, in 1x TBE buffer in a glass flask, and then swirling a bit. The polyacrylamide, ammonium persulfate, and TEMED can then be added.
2. Loading dye should contain xylene cyanol, bromophenol blue, 0.1% SDS, 93% deionized formamide, 80 ug/ml yeast RNA, and glycerol, and should be stored at -20 degrees C.
3. Heat samples at 93 degrees C for 3 min. in loading dye.
4. Run the gel using 1x TBE buffer as the running buffer until an adequate amount of separation between bands is achieved.
5. Visualize by staining the urea gel in ethidium bromide or SYBR Safe dye, or by transfering onto nitrocellulose similar to a Western blot and performing Northern blot analysis via standard procedures.