Have you ever wondered how many times you can use your electrophoresis buffer before replacing it? In this video, we'll explore the effects of electrophoresis on the pH of TAE buffer.

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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.

We use electrophoresis buffer for two main reasons. First, it adds charged ions to water to make the current flow through the gel, as water is a poor conductor of electricity. Secondly, the buffer keeps the samples at a biologically appropriate pH, which preserves their charge or structure.

pH is a measure of the acidity or alkalinity of a solution. pH values range from 0 to 14, with 7 being neutral, values less than 7 being acidic, and values greater than 7 being basic. 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 electrophoresis buffer.

The current is generated by a power supply that is connected to electrodes at either end of the electrophoresis chamber. When the power supply is turned on, the current moves the DNA through the agarose gel. However, the electricity also 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. These gasses form bubbles at the electrodes when we perform electrophoresis.

Because of these chemical reactions, the buffer at the cathode will become more basic, and the buffer at the anode will become more acidic. Over time, this will change the pH of the 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.

Online, people recommend using TAE buffer anywhere from one to ten times before replacing it. Generally, I use buffer two to three times before starting again fresh since TAE has a pretty low buffering capacity. That’s about 90 minutes of electrophoresis. So, let’s try an experiment. How long does it take to change the pH of our electrophoresis buffer? And therefore, how many times can we use our buffer?

We’re going to do a simple experiment. We’re going to fill our electrophoresis chamber with TAE buffer and add a universal pH indicator. We’ll attach the electrodes and turn on the power supply to start electrolysis of the buffer. As the pH changes, we should be able observe the color change of the indicator. More acidic solutions will become red or yellow, and more basic solutions will become indigo or purple.

What will we find? Be sure to watch the video to see our final recommendation!

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