Gel Electrophoresis- A Separation Technique
Have you ever wondered how is DNA fingerprinting done? It is done by involving a special step of gel electrophoresis. Gel electrophoresis is a technique generally used to separate charged molecules like DNA fragments, RNA, and proteins based on their charge and size. Let’s dig in deeper and learn the principle of gel electrophoresis.
The most common approach is agarose gel electrophoresis where agarose is obtained from algae sources like Gracilaria and Gellidium. The process of making agarose gel usually involves heating of agarose in a buffer. After cooling, it leads to formation of a solidified gel that is used in the process of gel electrophoresis. In simple terms, a gel can be considered as a matrix of agarose molecules that are held together particularly by hydrogen bonds. Specialised wells are present at the end of the agarose gel, the DNA sample is loaded in these wells.
Principle of gel electrophoresis:
The principle of gel electrophoresis is very simple. The DNA fragments are negatively charged molecules, thus they have a tendency to move towards the positive electrode called the anode. Hence, when electric current is made to run through the agarose gel, they are forced to move and get separated.
The smallest fragment of DNA will move the farthest.
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Purpose of using gel electrophoresis:
There are various purposes of gel electrophoresis:
- For visualizing the samples of DNA.
- For constructing recombinant DNA molecules.
- To find out whether DNA is present in a sample or not.
- To find out about the sizes of DNA fragments present.
- To find out about incomplete digestion or presence of contaminants like RNA in a sample.
- For separating the DNA fragments for purification.
Gel electrophoresis includes the following steps:
- Preparation of the running samples- first the DNA is isolated from a source. It is then combined with blue dye to visualize it.
- Preparation of an agarose TAE gel solution – during the process of electrophoresis is must, TAE buffer has the role to provide source of ions for settling up the electric field.
- Gel casting – the agarose TAE solution is then poured into a casting tray. It is allowed to cool down and solidify.
- Setting up the electrophoresis chamber- The gel is then placed in the chamber filled with TAE buffer.
- Gel loading– The DNA ladder as well as DNA sample is loaded in the wells.
- Electrophoresis– the positive and negative electrodes are connected with the gel electrophoresis chamber. The power supply is turned on which provides the electric field. The negatively charged DNA samples start to move slowly through the agarose gel. The pattern of movement can be understood from the principle of gel electrophoresis.
- Visualization of DNA- The gel is removed and then placed into ethidium bromide solution which tends to intercalate between DNA double helix and this is visible in UV light.
DNA bands are visualized in each lane. Length of DNA can be estimated with the DNA ladder that was loaded along with DNA samples.
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Watch how to load a sample for gel electrophoresis–https://www.instagram.com/reel/CcQuurXoKhF/?utm_source=ig_web_copy_link