One of the functions of buffers during electrophoresis is to maintain the proper pH. During electrophoresis, both the positive electrode and the negative electrode will undergo an electrolytic reaction. The positive electrode is an oxidation reaction (4OH--4e->2H2O+O2), and the negative electrode is a reduction reaction (4H++4e->2H2). The positive electrode becomes acidic and the negative electrode becomes alkaline. A good buffer system should have a strong buffer capacity, so that the pH of the solution at both poles remains basically unchanged.
Another function of the electrophoresis buffer is to make the solution have a certain conductivity to facilitate the migration of DNA molecules. For example, the general electrophoresis buffer should contain 0.01-0.04mol/L of Na+ ions. When the concentration of Na+ ions is too low, electrophoresis The speed becomes slower; if it is too high, it will cause excessive current to cause the glue to heat up or even melt.
Another component of the electrophoresis buffer is EDTA, which is added at a concentration of 1-2 mmol/L. The purpose is to chelate Mg2+ ions, prevent DNase activation during electrophoresis, and prevent Mg2+ ions and nucleic acids from forming precipitation.
TAE is the most widely used cushioning system. Its characteristics are that the supercoiled electrophoresis is more in line with the actual relative molecular mass (the relative molecular mass measured during electrophoresis in TBE will be greater than the actual molecular mass), and the mobility of double-stranded linear DNA in it is higher than that of the other two buffers. It is about 10% faster. When electrophoresing fragments larger than 13kb, TAE buffer will achieve better separation effect. In addition, TAE buffer system can also be used for electrophoresis when recovering DNA fragments. The disadvantage of TAE is that the buffer capacity is small, and it is not suitable for long-term electrophoresis (such as overnight) unless there is a circulation device to exchange the buffer between the two poles.
TBE is characterized by strong buffering capacity. TBE can be used for long-term electrophoresis, and the separation effect is better when it is used for electrophoresis of fragments smaller than 1kb. When TBE is used in agarose gel, it is easy to cause high electroosmosis, and the recovery rate of DNA fragments is reduced due to the interaction with agarose to generate non-covalently bound tetrahydroxyborate complexes, so it is not suitable for use in recovery electrophoresis.
The buffering capacity of TPE is also strong, but because phosphate is easily precipitated during ethanol precipitation, it is not suitable for electrophoresis to recover DNA fragments.