Introduction
Gel electrophoresis is a widely used technique that employs a gel matrix to facilitate the separation of macromolecules like DNA, RNA, and proteins.
The gel acts as a sieve, enabling high-resolution separation based on size and charge.
Principle
Molecules migrate through the porous gel matrix under an electric field, with smaller or more highly charged molecules moving faster than larger or less charged ones.
Methodology
Gel Preparation: A gel matrix (commonly agarose for nucleic acids or polyacrylamide for proteins) is prepared and poured into a casting tray with a comb to create wells.
Sample Loading: The sample mixtures are loaded into the wells.
Buffer Setup: An appropriate running buffer is added to the electrophoresis chamber to facilitate ion conduction.
Application of Electric Field: An electric current is applied, causing charged molecules to migrate through the gel.
Visualization: After electrophoresis, the gel is stained with specific dyes (e.g., ethidium bromide for DNA) and visualized under UV light or other imaging systems.
Advantages
High Resolution: Effective separation of closely related molecules.
Versatility: Suitable for DNA, RNA, proteins, and other macromolecules.
Quantitative Analysis: Allows for the estimation of molecular sizes and concentrations.
Reproducibility: Consistent results with standardized protocols.
Visualization: Clear band patterns facilitate identification and analysis.
Disadvantages
Complexity: More steps and careful handling compared to simpler methods.
Time-Consuming: Gel preparation and electrophoresis can be time-intensive.
Cost: Reagents, gels, and equipment can be relatively expensive.
Limited Throughput: Typically analyzes a limited number of samples simultaneously.
Hazardous Dyes: Use of toxic stains like ethidium bromide requires safety precautions.
Applications of Gel Electrophoresis
Genetic Analysis: DNA fingerprinting, restriction fragment length polymorphism (RFLP) analysis.
Protein Profiling: SDS-PAGE for protein separation and analysis.
RNA Studies: Northern blotting and RNA expression analysis.
Clinical Diagnostics: Identifying genetic disorders and infections.
Molecular Cloning: Verification of DNA insertions and plasmid constructs.
6. Below is a tabulated differentiation between vertical and horizontal gel electrophoresis
Parameter | Vertical Gel Electrophoresis | Horizontal Gel Electrophoresis |
|---|---|---|
Orientation | Vertical | Horizontal |
Gel Type | Typically polyacrylamide gels | Commonly agarose gels |
Sample Wells | Created at the top using a comb during polymerization | Created using a comb in the gel mold after solidification |
Equipment Design | Two glass plates held together with clips or a frame | Flat tray where agarose is poured and solidified |
Applications | Protein separation, DNA sequencing | DNA and RNA fragment separation like restriction digests or PCR products |
Handling | More cumbersome, risk of breaking glass or tearing gel | Easier to handle, manipulate, and transfer |