Agarose gel is a substance used in biochemistry and biotechnology for gel electrophoresis and size exclusion chromatography. It is made from various types of algae and is used to separate and analyze proteins and DNA based on their size and electrical charge. Agarose is extracted from red marine algae and can be used as a food thickener, laxative, or medium for the growth of microorganisms. The gel is structured in a way that allows molecules of different sizes to move through it at different speeds, making it useful for analyzing and isolating DNA sequences. It is also used in size exclusion chromatography to separate molecules based on their size.
Agarose gel is a substance used in biochemistry and biotechnology for gel electrophoresis and size exclusion chromatography, which are methods of selecting large molecules based on their size and electrical charge. These processes use agarose to separate and analyze proteins and DNA. It is very suitable for these applications due to its molecular structure, which allows molecules of different sizes to move through it at different speeds. The material is made from various types of algae, and is usually found in laboratories in the form of a powder. To create a suitable medium for a given test, the powder is added to water at the appropriate strength, boiled, and then allowed to cool into a gel.
Production
Agarose is extracted in the form of agar from several species of red marine algae, or kelp, found in California and East Asia. Agar, a term derived from the Malay word agar-agar, meaning jelly, is typically made from the Gelidium types of seaweed. It is made up of two different substances, known as agarose and agaropectin, and provides support to the cell walls within marine algae. Once removed, the agar can be used as a food thickener, just like gelatin, or as a laxative. If purified, it can be used as a medium for the growth of bacteria, fungi or other microorganisms.
It is quite easy to separate agarose from agaropectin in agar because agarose molecules bind strongly to each other, while agaropectin gels poorly. There are several methods of obtaining agarose isolation. In one method, carrageenan, another molecule found in red algae, and a salt are added to the agar. This causes the agaropectin to precipitate or form a solid that can be removed from the agar solution. Another method adds the enzyme pectinase, a chemical that breaks down agaropectin, allowing it to dissolve in water.
Gel electrophoresis
Agarose gel is most commonly associated with electrophoresis. In this procedure, scientists apply an electric field to a slab of material containing dissolved DNA, RNA or protein fragments. This causes these large molecules to move due to their electrical charges – the positively charged types will move to the negative side and vice versa. DNA and RNA fragments have a negative charge and therefore will drift towards the positive end, while protein fragments can be either negative or positive.
How fast molecules move depends on their size and how much charge they have. The agarose gel is structured in such a way that it forms a sort of network, with holes through which other molecules can travel. It is easier for the little ones to pass through the holes and therefore travel faster. Among the larger molecules, the shape also plays a role, as the more compact ones pass more easily. The technique is used both to analyze samples and to isolate particular DNA sequences for use in biotechnological applications.
Before electrophoresis, a DNA sample would be treated with special enzymes that cut the long strand-like molecules at specific points, creating smaller fragments. The agarose gel is prepared by dissolving the powder in a buffer solution, which resists changes in pH – acidity/alkalinity – which could otherwise result from electrochemical effects. Different amounts of powder are used for different ranges of molecules, but usually the concentration is between 0.7 and 1.2%. At this point, a fluorescent dye called ethidium bromide is usually added, as it stains the DNA and makes it easily visible under ultraviolet light. This mixture is then microwaved and allowed to solidify.
The DNA samples are placed in small wells in the gel and a direct electric current is applied through them. Molecules of different sizes travel through the gel at different speeds, so after a while they will appear in different positions, with the smallest fragments more towards the positive end. This allows scientists to determine fragment sizes and isolate different DNA sequences.
other uses
Agarose gel is sometimes used in a related technique that doesn’t involve electricity, known as size exclusion chromatography. In this method, a glass column is filled with gel beads and a solution containing molecules of different sizes is poured. In contrast to electrophoresis, larger molecules move faster down the column to emerge at the bottom, while the progress of smaller ones is slower in the beads. This is because small molecules tend to be absorbed into the pores of the gel, while larger ones are too large to enter these pores and tend to flow between the beads instead. The gel type and concentration can be adjusted to suit the size of the molecule to be separated.
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