What is agarose and agarose gel?
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What is Agarose?
Agarose is a polysaccharide extracted from seaweed, such as red algae, and its main component is galactose. Agar is composed of agar and agar pectin, which is purified to obtain agar. Agar is a linear polycrystalline, while agar pectin is a heterogeneous mixture of many smaller molecules. Agarose has a linear structure and consists of alternating D-galactose and 3,6-anhydrous galactose, alternating to form repeating disaccharide units by β-1,4 and α-1,3 bonds.
Gel properties
of agarose Agarose can be dissolved in heated water (at least 90°C or higher) and when the solution cools below 45°C, it forms a semi-solid gel. It is also a very good gel. The gelling properties of agarose are its main feature and the basis for its many uses. The properties of agarose gels are usually expressed in terms of gel strength. The higher the strength, the better the gel performance. The concentration of high-quality agarose is usually higher than 1200 g/cm2 (gel concentration 1%). The gelatinizing properties of agarose stem from the hydrogen bonds between agarose chains, and anything that may break the hydrogen bonds can lead to the destruction of gelatinization. Agarose is hydrophilic and virtually free of charged groups, virtually unchangeable and adsorpable-sensitive biomolecules, making it an ideal inert carrier. The gel temperature depends on the monomer composition (methoxy content) and solution concentration, as well as the chemical derivation of the polymer, such as hydroxyethyl derivation or the addition of harmful salts. The pore size of agarose gel depends on its content. Microspheres with an agarose content of 6% are usually used, with an average pore size of about 30 nanometers; while the microspheres with an agarose content of 4% and 2% have pore sizes of 70 nm and 150 nm, respectively. Agarose with larger diffusion pores is used in gel electrophoresis, allowing macromolecules such as DNA to pass through.
The Role of Agarose in Electrophoresis Techniques What is Electrophoresis Techniques?
Electrophoresis is a phenomenon in which a charged substance moves in an electric field towards an electrode opposite to its charge. Various biomolecules can dissociate into charged ions under specific pH conditions, which move towards opposite electrodes in an electric field. Electrophoresis is a very effective method for analyzing biological macromolecules such as nucleic acids and proteins. When a gel containing an electrolyte is in an electric field, the ions in it move, and their speed of movement varies depending on the size, shape, and charge of the ions. By exploiting differences in motion speeds, various molecules of different sizes can be distinguished, detected, and analyzed.
What is agarose gel electrophoresis?
Agarose gel electrophoresis is an electrophoresis method that uses agarose as a support medium. The main difference between its analytical principle and other electrophoresis is that it has the dual function of molecular screening and electrophoresis. Agarose gels have a network structure, and the material molecules are blocked by resistance when they pass through, so in gel electrophoresis, the separation of charged particles depends not only on the nature and quantity of the net charge, but also on the size of the molecules, which greatly improves the discrimination ability. However, due to its large pore size, its molecular sieving effect is almost negligible for most proteins and is now widely used in nucleic acid research. Proteins and nucleic acids have different charges depending on the pH and operate at different speeds due to different forces in the electric field, and can be separated according to this principle. The pH of the electrophoresis buffer is between 6 and 9, and the ionic strength is between 0.02 and 0.05 as optimal. 1% agarose is often used as an electrophoresis support. Agarose gels can distinguish between DNA fragments of about 100 base pairs. Although its resolution is lower than that of polyacrylamide gels, it is easy to prepare and has a wide range of separations. Plain agarose gels can separate 0.2 to 20 kb of DNA, and pulsed electrophoresis can separate DNA fragments up to 107 base pairs. The table below shows the optimal resolution range for linear DNA for different agarose gel concentrations.
However, RNA tends to form secondary structures, and sometimes there are multiple different species in the same fragment, which affects how it migrates. In this case, agarose native gels are usually not used to analyze the size of RNA, and alternative methods include RNA blotting and denaturing agarose gel electrophoresis, which utilize conditions capable of disrupting secondary structures.
Agarose Gel Electrophoresis
Reagent Agarose: Optionally free of detectable DNase/RNase, with the advantage of high intensity and low background.
Buffer: Triacetylethylenediaminetetraacetic acid (EDTA) (TAE) or triborate EDTA (TBE) is often the buffer of choice because the triacid solution is effective in buffering under slightly alkaline conditions, keeping DNA deprotonated and dissolved in water. EDTA is a chelating agent that inactivates nucleases that can damage the DNA being analyzed.
DNA staining: EB, GoldView Type I/II dyes, GelRed and GelGreen, SYBR Green, SYBR Gold, and SYBR Safe.
Agarose is a polysaccharide extracted from seaweed, such as red algae, and its main component is galactose. Agar is composed of agar and agar pectin, which is purified to obtain agar. Agar is a linear polycrystalline, while agar pectin is a heterogeneous mixture of many smaller molecules. Agarose has a linear structure and consists of alternating D-galactose and 3,6-anhydrous galactose, alternating to form repeating disaccharide units by β-1,4 and α-1,3 bonds.
Structure of agarose
Gel properties
of agarose Agarose can be dissolved in heated water (at least 90°C or higher) and when the solution cools below 45°C, it forms a semi-solid gel. It is also a very good gel. The gelling properties of agarose are its main feature and the basis for its many uses. The properties of agarose gels are usually expressed in terms of gel strength. The higher the strength, the better the gel performance. The concentration of high-quality agarose is usually higher than 1200 g/cm2 (gel concentration 1%). The gelatinizing properties of agarose stem from the hydrogen bonds between agarose chains, and anything that may break the hydrogen bonds can lead to the destruction of gelatinization. Agarose is hydrophilic and virtually free of charged groups, virtually unchangeable and adsorpable-sensitive biomolecules, making it an ideal inert carrier. The gel temperature depends on the monomer composition (methoxy content) and solution concentration, as well as the chemical derivation of the polymer, such as hydroxyethyl derivation or the addition of harmful salts. The pore size of agarose gel depends on its content. Microspheres with an agarose content of 6% are usually used, with an average pore size of about 30 nanometers; while the microspheres with an agarose content of 4% and 2% have pore sizes of 70 nm and 150 nm, respectively. Agarose with larger diffusion pores is used in gel electrophoresis, allowing macromolecules such as DNA to pass through.
An agarose gel plate
The Role of Agarose in Electrophoresis Techniques What is Electrophoresis Techniques?
Electrophoresis is a phenomenon in which a charged substance moves in an electric field towards an electrode opposite to its charge. Various biomolecules can dissociate into charged ions under specific pH conditions, which move towards opposite electrodes in an electric field. Electrophoresis is a very effective method for analyzing biological macromolecules such as nucleic acids and proteins. When a gel containing an electrolyte is in an electric field, the ions in it move, and their speed of movement varies depending on the size, shape, and charge of the ions. By exploiting differences in motion speeds, various molecules of different sizes can be distinguished, detected, and analyzed.
Electrophoresis instruments
What is agarose gel electrophoresis?
Agarose gel electrophoresis is an electrophoresis method that uses agarose as a support medium. The main difference between its analytical principle and other electrophoresis is that it has the dual function of molecular screening and electrophoresis. Agarose gels have a network structure, and the material molecules are blocked by resistance when they pass through, so in gel electrophoresis, the separation of charged particles depends not only on the nature and quantity of the net charge, but also on the size of the molecules, which greatly improves the discrimination ability. However, due to its large pore size, its molecular sieving effect is almost negligible for most proteins and is now widely used in nucleic acid research. Proteins and nucleic acids have different charges depending on the pH and operate at different speeds due to different forces in the electric field, and can be separated according to this principle. The pH of the electrophoresis buffer is between 6 and 9, and the ionic strength is between 0.02 and 0.05 as optimal. 1% agarose is often used as an electrophoresis support. Agarose gels can distinguish between DNA fragments of about 100 base pairs. Although its resolution is lower than that of polyacrylamide gels, it is easy to prepare and has a wide range of separations. Plain agarose gels can separate 0.2 to 20 kb of DNA, and pulsed electrophoresis can separate DNA fragments up to 107 base pairs. The table below shows the optimal resolution range for linear DNA for different agarose gel concentrations.
| Agarose concentration | Optimal linear DNA resolution range (bp) |
| 0.5% | 1,000-30,000 |
| 0.7% | 800-12,000 |
| 1% | 500-10,000 |
| 1.2% | 400-7,000 |
| 1.5% | 200-3,000 |
| 2.0% |
50-2,000 |
However, RNA tends to form secondary structures, and sometimes there are multiple different species in the same fragment, which affects how it migrates. In this case, agarose native gels are usually not used to analyze the size of RNA, and alternative methods include RNA blotting and denaturing agarose gel electrophoresis, which utilize conditions capable of disrupting secondary structures.
Agarose Gel Electrophoresis
Reagent Agarose: Optionally free of detectable DNase/RNase, with the advantage of high intensity and low background.
Buffer: Triacetylethylenediaminetetraacetic acid (EDTA) (TAE) or triborate EDTA (TBE) is often the buffer of choice because the triacid solution is effective in buffering under slightly alkaline conditions, keeping DNA deprotonated and dissolved in water. EDTA is a chelating agent that inactivates nucleases that can damage the DNA being analyzed.
DNA staining: EB, GoldView Type I/II dyes, GelRed and GelGreen, SYBR Green, SYBR Gold, and SYBR Safe.
Gel recovery kit: It is mainly used to recover DNA of different lengths from various agarose gels, and DNA recovery from gels can be completed within 15-30 minutes, and the recovery efficiency is extremely high.
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