1. During the run of the glyoxal gel, measure gel exactly and set up for transfer to nitrocellulose (step 2). After run, cut off bottom right corner of gel. The gel may be transferred immediately after electrophoresis is complete. It is not necessary to place the glyoxal gel in 20X SSC before transfer.
• If the gel is > 5 mm thick, or is >1% agarose, or the RNA to be analyzed is > 2.5 kb, then:
A. Soak gel for 15 minutes in freshly made 50 mM NaOH at room temperature.
B. Wash gel in DEPC treated ddH2O.
C. Soak gel for 45 minutes in 20X SSC at room temperature. Then do transfer.
2. Cut nitrocellulose membrane about ~1 mm shorter than gel. Wet in membrane ddH2O for 5 minutes. Soak membrane 5-10 minutes in 20X SSC. Cut 2 sheets of THIN filter paper ~1 mm shorter than nitrocellulose. Cut 2 sheets of THICK paper ~1 mm shorter than nitrocellulose. Cut 2-3 inch stack of brown paper towels.
3. Set up Transfer. Use 2 pieces of thin filter paper to make wick. Place wick over glass plate on support platform. Remove air bubbles at all steps in the set up procedure. Put gel face down on wick. Put wetted nitrocellulose paper down correctly first time. Put on thin paper pieces that have been wet in 20X SSC. Assemble transfer as depicted below. Use 50-100 g weight on top. Glass plates work well. Place plastic wrap around gel to prevent evaporation.
4. After transfer, wash filter 5 minutes in 2X SSC. Remove excess liquid and dry blot with 3MM paper. Air dry for 30 minutes.
5. Bake at 70℃ in vacuum oven for 2 hours. Blot can be stored dry wrapped in foil.
6. Remove glyoxal by treating blot for 5 minutes with 20 mM Tris, pH 8 at 65℃. For this, prewarm buffer to 70℃ and pour in tray with blot. Place tray in oven preheated to 65℃.
7. Float membrane in 5X SSC and submerge for 2 minutes to wet. Proceed with hybridization.
