Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis.

BENCHMARKS It has been nearly three decades since the invention of DNA electro-phoresis (1−6). Current conductive media for DNA electrophoresis remain largely restricted to legacy Tris-acetic acid-disodium EDTA (TAE) and Tris-boric acid-disodium EDTA (TBE) at substantial ionic strengths, leading to higher cost and excessive heat generation , and limiting the voltage and speed of electrophoretic runs. TAE and TBE usually contain between 40 to 90 mM Tris, corresponding anion concentrations , and trace amounts of different forms of EDTA (1−2 mM) (7). Investigators have compared and analyzed TAE and TBE buffers in DNA electrophoresis; however, to our knowledge no one has substantially investigated the simplification and substitution of components of these buffers to achieve a more efficient and inexpensive conductive medium for DNA electrophoresis (8,9). It is well established that heat generation is a primary source of problems in TAE and TBE gels, causing sample diffusion, convection, denaturation, and poor gel integrity, and limiting the ability to run gels at a high voltage (10). Ohm's law and the power law interrelate voltage (V), current (I), and power (P) (11,12). Power consumed in the elec-trophoresis system manifests as heat generation (P = VI). These interrelated variables are affected by ionic conduc-tance due to choice of salts and ionized components in proportion to their particular concentrations in the media used in electrophoresis. A thorough analysis of these critical properties of widely used buffered media for DNA electrophoresis was performed. We found that TAE and TBE create a " runaway " positive feedback loop of current and temperature, which results in poor gel resolution at high voltage (Figure 1, lanes 3−4). This positive feedback loop makes low-voltage (5−10 V/cm) runs necessary and highlights the limitations of using Tris as a cation for DNA electrophoresis. Another component of currently used buffers, EDTA, is now largely superfluous , since most DNA samples are readily soluble and since commonly used enzymes today would not carry an undesirable enzymatic activity under electrophoretic conditions. We thereby explored alternative, low-molarity, sodium-based conductive media that could mitigate this feedback loop. Extensive studies established that sodium concentrations between 7.5 and 12.5 mM, with borate as the counter-ion, provided the best resolution (data not shown). We therefore prepared a sodium boric acid (SB) conductive medium, where 1× SB consisted of 5 mM disodium borate decahydrate or 10 mM sodium hydroxide, pH adjusted to 8.5 with boric acid. A 20× stock solution of …