Electrophoresis is a method used to separate fragments of DNA or RNA based on their size (length*) using an electric current. It has applications in processes such as genetic fingerprinting and disease diagnosis. DNA and RNA are negatively charged which means they will move towards a positive electrode. This technique can be used for proteins, but as the charge is variable in proteins they are first mixed with a chemical to give them all the same negative charge. In this article, we will focus on DNA electrophoresis.
*Note to A-level biology students: be careful to find out whether your specification like you to talk about size or length in relation to this topic.
Preparing electrophoresis equipment
Electrophoresis equipment consists of a gel tank and a gel tray. The gel tank is filled with a buffer solution and is connected to a power supply. The gel tray is used to prepare an agarose gel as follows:
- An agarose solution is prepared.
- The solution is poured into the gel tray and left to solidify* into a gel.
- The tray is placed into the tank and covered with buffer solution. The wells must be at the negative electrode end of the tank as the DNA will move towards the positive electrode.
*While the gel is solidifying, a comb is left in to ensure that the gel contains wells along one end. Why not watch this short video to see the process in action.
Loading DNA into the agarose gel
This is a fiddly process which requires a lot of practise. Firstly, the solution containing the DNA fragments must be mixed with loading dye. This is to help the DNA sink to the bottom of the wells. Then a pipette is used to transfer about 10μl of the mixture into a well (very carefully to avoid making a hole in the gel or missing the well altogether). This is then repeated for all the samples, but must be done quite quickly so that the first sample hasn’t diffused away by the time you finish!
Running the electrophoresis
Once all the samples are loaded, the electric current can be switched on. DNA is negatively charged so will move towards the positive electrode. Shorter fragments will travel more quickly and therefore move further through the gel over the the time. Once the loading dye has nearly reached the end of the gel, the electric current is switched off and the gel is removed from the tank. However, the DNA fragments are colourless and cannot be seen yet.
Visualising the DNA fragments
Various different staining options are available for electrophoresis gels, but most require the gel to be viewed under UV light. For example, a fluorescent tag can be added to the fragments before loading into the wells, or a chemical called ethidium bromide can be used.
Sometimes it is helpful to run a DNA ladder in one of the lanes – this is a solution containing DNA fragments of known sizes which can be used to help estimate the size of your fragments.
- An agarose gel is prepared and loaded into a gel tank connected to a power supply.
- DNA fragments are mixed with loading dye and pipetted into the wells.
- The electric current is switched on. Smaller DNA fragments will travel further through the gel because they move more quickly.
- The DNA must be stained and viewed under UV light.
Leave a Reply