Control of buffer pH during agarose gel electrophoresis of glyoxylated RNA.

There is a shift in buffer pH routinely encountered during electrophoresis. If one is running glyoxylated RNA on agarose gels, this pH shift can have potentially detrimental effects on experimental objectives if the shift is allowed to proceed unabated; this is due to the fact that the RNA will deglyoxylate if the pH is allowed to rise above 8.0. In order to counteract the shift, the buffer may be continuously recirculated or totally replaced at predetermined intervals. Because constant recirculation may be technically bothersome to achieve and because repeated total buffer replacement may require large amounts of highly purified water, we have studied (a) the time course of the pH change encountered during the electrophoretic process and (b) whether or not simple manual mixing of the buffer system at specific intervals was sufficient to maintain pH within acceptable bounds. We found that, under the given conditions, the cathode pH had nearly reached 8.0 at 25 min and had soared to 10.5 at 40 min. We further found that manual mixing at 20-min intervals not only prevented the cathode pH from rising above 7.8, but also restored the buffer to its initial pH.