In genetics, gel electrophoresis is a lab technique employed to separate mixtures comprising DNA, RNA, and other proteins based on their specific molecular size and charge. To carry out this method, the DNA, RNA, or proteins to be separated are passed through a gel containing tiny pores. An electric field propels the molecules through the gel.
Horizontal gel electrophoresis separates DNA, RNA, or protein molecules based on their molecular size and charge, using a continuous buffer-immersed gel in a horizontal orientation. The gel box is divided into two compartments by an agarose gel, with an anode at one end and a cathode at the other. Applying a current in this technique creates a charge gradient facilitated by the buffer. When a load is applied, the gel heats up, but the buffer acts as a coolant, maintaining optimal temperature levels. Recirculating the running buffer prevents pH gradient formation. Since the gel system's two compartments connect with the running buffer, a discontinuous buffer system cannot be used. Oxygen exposure in the gel box prohibits the use of acrylamide in horizontal gel electrophoresis. Oxygen inhibits acrylamide polymerization, interfering with gel formation. Horizontal gel electrophoresis is a straightforward method for separating DNA and RNA molecules.
To start gel electrophoresis, you will require a set of essential tools. Although there are various types of gel electrophoresis, the necessary equipment remains consistent. Here is a list of what you'll need to initiate gel electrophoresis:
The gel box used in gel electrophoresis varies depending on the type of gels being utilized. For DNA and RNA separation with agarose gels, horizontal gel electrophoresis is the preferred choice.
The gel used in gel electrophoresis can either be pre-cast or hand-cast. It contains wells at the top where the samples are loaded before they migrate through the gel. The gel is immersed in a running buffer that contains salt ions, which facilitate the conduction of electrical current throughout the gel.
Beforehand, conduct research to determine the specific running buffer required and make preparations accordingly. Running buffers can be obtained through purchase, but they can also be produced in larger quantities within the laboratory.
To supply the necessary electrical current, a power supply such as the Denagene Tajhiz Power Supply is utilized. Cables are connected to the positive and negative terminals of the gel box, allowing the power supply to deliver the electrical current.
Each experiment has different samples, but they all need to be prepared in a similar way before doing gel electrophoresis. To start gel electrophoresis, mix your samples with a loading buffer. The loading buffer has dye to help you see the sample while loading and running it, and glycerol to make the samples denser. Making the samples denser helps them sink to the bottom of the wells during loading, which stops the light samples from spreading out too fast.
You can either buy ready-made gels (pre-cast) or make them yourself in the lab (hand cast). When preparing the gel, there are several factors to consider, such as the gel composition, the percentage of the gel (which affects pore size and separation resolution), the number of wells needed, and the size of those wells.
Choose the appropriate type of running buffer and prepare it in advance. Buffers can be purchased, but they are often made in large quantities in the lab. When you're ready to load, take out the comb from the gel. Fill the gel box with the running buffer and place the gel in the box so that it is completely covered by the running buffer.
Before adding the samples, decide on the best order for placing them on the gel. Use a pipette to carefully place the samples into individual wells on the gel. Additionally, include a ladder with specific size markers in one of the wells as a reference for future analysis.
Once the samples are loaded, cover the gel box with the lid, connect the cords to the power supply, and run the gel using electrophoresis. Adjust the voltage and duration based on the specific requirements of your lab's experiment.
After completing the steps for gel electrophoresis, it's important to visualize the resulting bands of DNA, RNA, or protein. DNA gels can be visualized by adding a DNA stain to the gel and examining it under UV light. Additional steps are necessary for visualizing DNA and RNA blots. Proteins can be visualized within the gel using techniques like two-dimensional difference gel electrophoresis (2D-DIGE), but they often need to be transferred from the gel to a membrane for further analysis. Digital imagers such as the Azure Imagers enable both visualization and documentation of results in a single, efficient step.