Denagene Tajhiz
Issue of date and time
Time To Read Minute
Writer

How Does Horizontal Gel Electrophoresis Operate?

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.

What is Horizontal gel electrophoresis? 

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.

What Equipment and Materials Are Essential for Conducting Gel Electrophoresis?

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:

Gel Box

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. 

Gel

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.

Running buffer

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.

Power supply

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.

What Are the Horizontal Gel Electrophoresis Steps? 

Phase 1: Get the sample ready 

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.

Phase 2: Get the gel and buffer ready

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.

Phase 3: Load and pipette the samples

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.

Phase 4: Perform electrophoresis (Run the gel)

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.

Phase 5: Visualize and record the bands

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.

FAQ
1. How does horizontal gel electrophoresis work?
Horizontal gel electrophoresis involves casting a gel horizontally and placing it in a chamber divided into two sections. The gel is positioned between a positive charge at one end and a negative charge at the other, created by a continuous running buffer. When an electric current is applied, the charged molecules in the samples migrate through the gel towards the opposite charge, separating based on their size and charge.
2. What is the purpose of the running buffer in horizontal gel electrophoresis?
The running buffer in horizontal gel electrophoresis serves multiple purposes. It provides conductivity for the electric current to flow through the gel, maintains a stable pH environment, and helps dissipate heat generated during the electrophoresis process. Additionally, it assists in the migration of the charged molecules through the gel.
3. How are the samples loaded into the gel in horizontal electrophoresis?
In horizontal electrophoresis, the samples are loaded into the wells of the gel. Typically, a pipette is used to carefully and precisely place the samples into the wells. The samples are mixed with a loading buffer, which contains dye for visualization and glycerol to increase sample density, facilitating their sinking to the bottom of the wells.
4. What factors affect the separation of molecules in horizontal gel electrophoresis?
Several factors influence the separation of molecules in horizontal gel electrophoresis. The composition and percentage of the gel affect the pore size and, consequently, the separation resolution. The electric field strength, determined by the voltage and duration of the electrophoresis run, also impacts the separation. Additionally, the molecular size and charge of the molecules being analyzed play a significant role in their migration and separation within the gel.
5. How are the results visualized in horizontal gel electrophoresis?
After the electrophoresis run, the results are visualized to analyze the separated molecules. For DNA gels, a DNA stain is often used, which binds to the DNA molecules and allows their visualization under UV light. In the case of protein separation, special staining techniques or transfer to a membrane for subsequent analysis, such as Western blotting, may be required to visualize the protein bands accurately.
Customer's Comments
Submit a New Comment
    Your Message Was Successfully Submitted
    Subscribe to the Denagene Tajhiz Newsletter, you will receive the latest updates from the Denagene Tajhiz Company, such as new product information, special price offers, or competitions by e-mail. Unsubscribe at any time to tailor your preferences.
      Your Email has been successfully submitted
      Copyright 2024 Denagene Tajhiz Inc. All rights reserved