Looking at Bands from Another Side

Abstract

In high-percentage gels run in the submerged electrophoresis mode separated DNA bands were bent, regardless of whether the gel was prepared from polyacrylamide, poly-N-acryloyl-tris(hydroxymethyl)aminomethane, or hydroxyethylated agarose. This declination from the vertical axis is the major reason for the appearance of diffuse bands after recording by a camera positioned vertically above the gel. Five major factors which could cause the bending, including an inhomogeneous electric field, electroendosmosis, nonuniform heat dissipation, different effective porosity across the gel thickness, and different conductivity in the gel and electrophoresis buffer, were investigated. The results indicate that the major cause is the difference in the ratio of current density and specific conductivity between the gel and the running buffer. When that ratio was adjusted by empirically optimizing gel ionic composition, the bending could be prevented or greatly diminished. Other results described in this work suggest that electrophoretic systems in which gels are polymerized and run in the same buffer, frequently referred to as "continuous buffer systems," may not provide a continuous, constant electric field. Furthermore, the pH change regularly observed in circulated running buffers could be related to difference in the amount of current transported by buffer anions and cations. A buffer in which current was equally transported by anions and cations showed no detectable pH change after prolonged electrophoresis.