Agarose gel electrophoresis is a fundamental technique in molecular biology used to separate and analyze macromolecules, most commonly DNA and RNA. The process involves placing samples into a gel matrix and applying an electrical current to drive the molecules through its pores. This electrical force, specifically the voltage, initiates and sustains the separation. The technique separates molecules primarily based on size, with smaller fragments navigating the gel matrix more easily than larger ones.
How Voltage Drives Electrophoresis
The movement of nucleic acids through the gel matrix is dependent on the electric field generated by the applied voltage. DNA and RNA possess an inherent negative charge due to the phosphate backbone, causing them to migrate away from the negative electrode (cathode) and toward the positive electrode (anode) when current is applied.
The voltage setting dictates the strength of this electric field, which determines the speed at which the charged molecules move. Applying a higher voltage increases the electrophoretic force, causing the molecules to travel faster across the gel. This accelerated movement shortens the run time but introduces issues related to heat and resolution.
Key Factors Influencing Voltage Selection
Selecting the appropriate voltage setting is determined by several experimental factors. The concentration of agarose in the gel is a primary consideration because it controls the effective pore size of the matrix. Gels with a higher percentage of agarose have smaller pores, which increases resistance and necessitates a lower voltage to maintain good resolution.
The size of the target molecules being separated also influences the voltage choice. To effectively resolve large DNA fragments, a lower voltage is often preferred to allow the molecules to disentangle and move through the matrix without distortion. Conversely, when separating very small fragments, a slightly higher voltage can be used to accelerate the run, provided the resulting heat is managed.
Another significant variable is the type of electrophoresis buffer used, such as Tris-Acetate-EDTA (TAE) or Tris-Borate-EDTA (TBE). These buffers differ in their ionic strength. TBE has a higher ionic strength than TAE, which increases the overall conductivity of the system.
Higher conductivity means that the same voltage will generate more current and thus more heat. Consequently, gels run with TBE buffer often require a slightly lower voltage setting compared to those run with TAE to prevent overheating. The physical distance between the cathode and anode electrodes also dictates the voltage applied, as the field strength is a function of voltage divided by distance.
The Impact of Incorrect Voltage Settings
Operating an agarose gel at a voltage that is too high is the most common cause of poor separation. The passage of electrical current through the conductive buffer and gel matrix generates heat, known as Joule heating. Excessive voltage dramatically increases the rate of heat generation, which negatively affects the integrity of the gel and the quality of the separation.
This heat can cause the agarose matrix to partially melt, particularly toward the edges of the gel where cooling is less efficient. The uneven melting leads to distorted DNA migration, resulting in a characteristic “smiling” effect where bands curve upward at the sides. High heat also accelerates the breakdown and depletion of the running buffer, causing shifts in the pH and ionic strength, which further compromises DNA mobility and leads to band smearing or streaking across the lane.
Setting the voltage too low avoids heat problems but introduces issues related to time and diffusion. A low voltage results in an excessively long run time, allowing the separated DNA fragments more opportunity to spread out within the gel matrix. This molecular dispersion, or diffusion, causes the DNA bands to become broad and fuzzy. The reduced resolution makes it difficult to accurately determine fragment size or distinguish between fragments of similar length.
Calculating and Applying Optimal Voltage
In laboratory practice, the optimal voltage is expressed as a voltage gradient standardized per centimeter of electrode distance. This standard measure, the volts per centimeter (V/cm) rule, accounts for the physical dimensions of the electrophoresis unit. The distance used for this calculation is the straight-line measurement between the cathode and anode electrodes, not the length of the gel itself.
For most standard agarose gel separations, the recommended voltage gradient falls within a range of 5 V/cm to 10 V/cm. For example, if the distance between the electrodes in the gel box is 10 cm, a common setting would be 70 V (7 V/cm), which provides a good balance between speed and resolution. Higher V/cm settings are reserved for the separation of very small DNA fragments or when a rapid, lower-resolution result is acceptable. Once the power supply is connected, never touch the buffer or the gel while the unit is actively running.