Electrophoresis is a biotechnology technique that separates mixtures of molecules based on charge, shape, and size using electricity and a porous gel matrix. It is commonly used to separate dyes, proteins, and nucleic acids like DNA and RNA. During electrophoresis, the buffer plays two important roles – it adds charged ions to water to make the current flow through the gel, and it keeps the samples at a biologically appropriate pH, which preserves their charge or structure. But how many times can we reuse the electrophoresis buffer? In this Biotech Basics post, we hope to shed some light on the topic.
As you remember from our previous post, pH is a measure of the acidity or alkalinity of a solution. At a pH of around 8, DNA maintains its negative charge, which is needed for it to be propelled through the gel by the electrical current. That’s the pH of our TAE buffer. As the electricity runs through the buffer during electrophoresis, it splits the water molecules in the buffer into hydrogen and oxygen in a process called electrolysis. At the negatively-charged cathode, hydrogen ions are reduced, forming hydrogen gas. At the positively-charged anode, water is oxidized, creating electrons, hydrogen ions, and oxygen gas. Over time, these chemical reactions change the pH of the buffer solution and reduce its buffering capacity, or the ability of a buffer to neutralize acid or base. In turn, this affects both how DNA migrates through a gel, and its stability.
The number of times you can reuse electrophoresis buffer depends on the buffering capacity of the buffer and how quickly the pH changes during electrophoresis. At Edvotek, we generally recommend using the buffer two to three times before replacing it, but some online sources suggest the TAE buffer can be used up to 10 times! So we decided to test the buffering capacity of our TAE buffer by performing a simple experiment. We filled an electrophoresis chamber with TAE buffer and added a universal pH indicator. As the pH of the buffer changes, we expect that the pH indicator will change color.
Within the first thirty minutes of electrophoresis, the solution at the cathode became basic, while the anode buffer became acidic, as shown in the video to the right. Over the next hour, the pH changes intensified until the buffer was exhausted at the end of two hours. After the experiment, we determined the overall pH by mixing the buffer in the chamber and comparing its color to the starting color of the electrophoresis buffer. The starting buffer is an emerald green color, indicating our starting pH of 8. The final buffer is more of a sky blue color, indicating a pH of 10 or 11. At this pH, the high concentration of hydroxide ions can break the hydrogen bonds between two DNA strands, resulting in its denaturation. This could affect downstream uses of the DNA in applications like cloning or sequencing.
Based this experiment, TAE buffer can safely be reused twice, for two thirty-minute gel runs, before it needs to be replaced, as a result of TAE’s low buffering capacity. – for two thirty minute gel runs – without much of a problem. Anything longer than that, you should supplement with some fresh TAE or, better yet, dump out the chamber and start with fresh buffer.
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