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Loading DNA Samples onto an Agarose Gel for Electrophoresis - Image Copyright: science photo, Image ID: 214331152 via Shutterstock.com Principles of gel electrophoresisThe gel electrophoresis technique exploits the difference in size and charge of different molecules in a sample. The DNA or protein sample to be separated is loaded on to a porous gel placed in an ionic buffer medium. On application of electric charge, each molecule having different size and charge will move through the gel at different speeds. The porous gel used in this technique acts as a molecular sieve that separates bigger molecules from the smaller ones. Smaller molecules move faster across the gel while the bulkier ones are left behind. The mobility of the particles is also controlled by their individual electric charge. Two oppositely charged electrodes that are part of the system pull molecules of towards them on the basis of their charge. DNA gel electrophoresis lab demo How does it work?The gel used in gel electrophoresis is usually made of a material called agarose, which is a gelatinous substance extracted from seaweed. This porous gel could be used to separate macromolecules of many different sizes. The gel is submerged in a salt buffer solution in an electrophoresis chamber. Tris-borate-EDTA (TBE) is commonly used as the buffer. Its main function is to control the pH of the system. The chamber has two electrodes – one positive and another negative - at its two ends. Samples that need to be analyzed are then loaded into tiny wells in the gel with the help of a pipette. Once loading is complete, an electrical current of 50–150 V is applied. Now, charged molecules present in the sample start migrating through the gel towards the electrodes. Negatively charged molecules move towards the positive electrode and positively charged molecules migrate towards the negative electrode. The speed at which each molecule travels through the gel is called its electrophoretic mobility and is determined mainly by its net charge and size. Strongly charged molecules move faster than weakly charged ones. Smaller molecules run faster leaving behind the larger ones. Thus, strong charge and small size increases a molecule’s electrophoretic mobility, while weak charge and large size decreases the mobility of a molecule. When all molecules in a sample are of the same size, the separation will solely be based on their size. Once the separation is complete, the gel is stained with a dye to reveal the separation bands. Ethidium bromide is a fluorescent dye commonly used in gel electrophoresis. The gel is soaked in a diluted ethidium bromide solution and then placed on a UV transilluminator to visualize the separation bands. The bands are immediately examined or photographed for future reference, as they will diffuse into the gel over time. The dye can also be loaded into the gel well in advance to track the migration of the molecules as it happens. Applications of gel electrophoresisGel electrophoresis is widely used in the molecular biology and biochemistry labs in areas such as forensic science, conservational biology, and medicine. Some key applications of the technique are listed below:
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Last Updated: Aug 23, 2018 Written by Susha CheriyedathSusha has a Bachelor of Science (B.Sc.) degree in Chemistry and Master of Science (M.Sc) degree in Biochemistry from the University of Calicut, India. She always had a keen interest in medical and health science. As part of her masters degree, she specialized in Biochemistry, with an emphasis on Microbiology, Physiology, Biotechnology, and Nutrition. In her spare time, she loves to cook up a storm in the kitchen with her super-messy baking experiments. CitationsPlease use one of the following formats to cite this article in your essay, paper or report:
Suggested ReadingWhy is gel electrophoresis an important tool used in biotechnology?In molecular biology, this technique is used to separate biological compounds, such as DNA or proteins, based on their size. Like the colored dyes, DNA and proteins are negatively charged, so they will migrate towards the positive electrode at different speeds depending on their size.
What is a gel electrophoresis used for?Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.
How electrophoresis is used in biotechnology?Electrophoresis is a laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge. An electric current is used to move the molecules through a gel or other matrix.
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Gel electrophoresis is an important tool used in biotechnology for forensics and paternity test.
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