Agarose gel is a widely used material in molecular biology laboratories, primarily serving as a medium for separating large biological molecules. This separation technique, known as gel electrophoresis, allows researchers to analyze DNA, RNA, or proteins based on their size and charge. The gel provides a porous matrix through which these molecules travel when an electric current is applied. Its straightforward preparation and effectiveness across a range of molecular sizes contribute to its widespread adoption.
The Primary Ingredient: Agarose
The fundamental component of agarose gel is agarose, a naturally occurring polysaccharide polymer. This complex sugar molecule is primarily extracted from certain species of red algae, such as Gelidium and Gracilaria, which are harvested from marine environments. The process begins with boiling the raw algae to extract agar, a substance that contains both agarose and a less purified component called agaropectin. Agarose is then purified from agar by removing the agaropectin.
This purification yields a high-purity agarose powder, which is crucial for consistent and reliable laboratory results. Chemically, agarose is a linear polymer composed of repeating disaccharide units called agarobiose. Each agarobiose unit consists of D-galactose and 3,6-anhydro-L-galactopyranose, linked together in a specific arrangement. This precise molecular structure is what gives agarose its unique gelling properties, making it an ideal matrix for molecular separation.
The Supporting Liquid: Buffer Solution
Agarose powder is dissolved in a specific buffer solution to create the gel. The buffer maintains a stable pH throughout the electrophoresis process. A consistent pH is important because it ensures the molecules being separated retain their charge and migrate predictably through the gel.
Commonly used buffers for preparing agarose gels include Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE). These buffers contain Tris base, which acts as a buffering agent, combined with either acetic acid or boric acid to establish the necessary pH. Both TAE and TBE buffers incorporate ethylenediaminetetraacetic acid (EDTA), a chelating agent. EDTA binds to metal ions, thereby protecting nucleic acids from degradation by enzymes that require these ions for their activity.
How These Components Form a Gel
To form the gel, agarose powder is dissolved in the buffer solution by heating it to near-boiling temperatures (typically 90-100°C). This heating process causes the agarose polymers to uncoil and disperse evenly throughout the buffer, forming a homogeneous solution. The solution is then allowed to cool, typically solidifying at temperatures between 35-42°C, depending on the specific agarose type and concentration.
As the solution cools, individual agarose polymer chains begin to associate. They form helical structures that then aggregate into larger, supercoiled bundles. These bundles are held together by extensive hydrogen bonding, creating a three-dimensional mesh-like network. This intricate network defines the gel’s structure, forming interconnected channels and pores essential for separating molecules.
Why These Materials Are Chosen
Agarose is chosen for gel formation due to its ability to create stable gels with a wide range of pore sizes. The size of these pores can be controlled by adjusting the concentration of agarose in the gel, allowing for the separation of molecules across a broad size spectrum. This property makes agarose particularly well-suited for separating large macromolecules like DNA fragments, which vary significantly in length.
Agarose is highly valued for its non-toxic and inert nature. It interacts minimally with the biological molecules being separated, such as DNA and proteins, which helps prevent unwanted binding or denaturation. The buffer solution is selected to complement agarose by maintaining optimal conditions. It ensures electrical conductivity for molecular movement and protects the integrity of the sample, reinforcing the gel’s utility in precise molecular analysis.