Agarose gel serves as the primary medium for separating nucleic acid fragments, such as DNA and RNA, based on their molecular size. This matrix is a polysaccharide, a linear carbohydrate polymer purified from certain types of red algae, which forms a three-dimensional network when dissolved and cooled. The process of gel electrophoresis involves applying an electric current to move the negatively charged nucleic acid molecules through the microscopic pores of this gel toward a positive electrode. Creating this medium requires careful preparation to ensure the resulting gel has the precise physical characteristics needed to resolve the target molecules effectively.
Determining the Correct Concentration
The physical characteristic of the gel that determines its separating power, known as the resolving power, is directly controlled by the concentration of the agarose powder used. This concentration is typically expressed as a weight-to-volume percentage, indicating the grams of agarose per 100 milliliters of liquid buffer. The standard range for most laboratory applications falls between 0.5% and 2.0%.
The percentage chosen is inversely related to the size of the pores formed within the gel matrix. A lower percentage results in a loose mesh with larger pores, which is necessary for allowing very large DNA fragments, often those greater than 1,000 base pairs, to pass through the gel efficiently. For example, a 0.7% to 1.0% gel is typically selected when the goal is to resolve these larger molecules.
Conversely, a higher percentage of agarose creates a much denser matrix with smaller pores. This tighter structure provides greater friction and sieving action, which is required to separate smaller DNA fragments, generally those under 500 base pairs, with greater precision. Gels in the 1.5% to 2.0% range are preferred for this high-resolution separation of small molecules.
Essential Materials and Buffer Preparation
The foundational components for creating the gel include:
- Pure agarose powder
- A suitable electrophoresis buffer
- A glass flask
- A precision scale
- A microwave or hot plate
- A gel casting tray
- A comb
The electrophoresis buffer is particularly important because it serves as the solvent for the agarose and, later, as the conductive medium for the electric current during the separation. It must be prepared before the agarose is added and dissolved.
Two common buffer types are Tris-Acetate-EDTA (TAE) and Tris-Borate-EDTA (TBE). TAE is used for separating larger DNA fragments or when DNA recovery is needed, due to its low ionic strength and faster migration rate. TBE offers a higher buffering capacity and provides sharper band resolution, making it the preference for separating smaller fragments.
Both buffer types are typically prepared from a concentrated stock solution, such as 10X or 50X, which must be diluted to the final working concentration, usually 1X, with deionized water. Using the correct working concentration ensures that the electrical resistance and pH remain stable throughout the electrophoresis run. The EDTA component in both buffers acts as a chelating agent to bind metal ions, which helps prevent the degradation of the nucleic acids by certain enzymes.
The Step-by-Step Gel Creation Process
The hands-on process begins with accurately weighing the calculated amount of agarose powder using the precision scale, based on the desired percentage and final volume of the gel. This powder is then transferred into an appropriately sized glass flask, and the prepared 1X electrophoresis buffer is added. The mixture should be swirled gently to suspend the powder before heating.
The flask is then heated, typically in a microwave oven, in short bursts until the solution is completely clear and all agarose particles have dissolved. Watch the flask carefully, as boiling over alters the final gel concentration due to buffer evaporation. Swirl the dissolved solution gently after heating to ensure homogeneity.
The molten agarose must then be allowed to cool significantly, usually to 50 to 60 degrees Celsius. Cooling prevents the comb and casting tray from warping and ensures that any necessary DNA binding stain, such as Ethidium Bromide or SYBR Green, is not degraded by excessive heat. The stain is added at this stage and mixed carefully to distribute it evenly.
The cooled, liquid agarose is then poured into the casting tray, which has been sealed and fitted with a comb to create the sample wells. Pour the solution slowly to avoid creating air bubbles and to form a gel of consistent thickness, usually around 5 millimeters. The gel is left undisturbed at room temperature for 15 to 30 minutes until it has fully solidified into a firm, opaque structure. Once solid, the comb is carefully removed straight up, leaving clean wells for sample loading, and the gel is ready for use.