In molecular biology, agarose gel electrophoresis is a frequently employed method for size-based protein, DNA, and RNA separation. Using the characteristics of agarose, a polysaccharide that is isolated from seaweed, this technique creates a gel matrix that allows proteins or nucleic acids to move across it in response to an electric field. This is a thorough breakdown of the procedure.
Table of Contents
Definition of Agarose Gel Electrophoresis
Using an electric field to a gel matrix consisting of agarose, a polysaccharide produced from seaweed, allows for the separation of DNA, RNA, or proteins according to their size and charge. This process is known as agarose gel electrophoresis.
Agarose gel electrophoresis works on the basis of charged molecules migrating across an agarose gel matrix when subjected to an electric field. Because of their phosphate backbone, DNA and RNA have a negative charge and move in the direction of the positive electrode. Size-based separation is possible because smaller molecules go through the gel pores more quickly and extensively than bigger ones.
Parts of Agarose Gel Electrophoresis
1. Agarose gel
A medium for molecular migration. The pore size is determined by the agarose content.
2. Electrophoresis Buffer
pH-maintaining, electrically conducting solutions like TBE (Tris-borate-EDTA) or TAE (Tris-acetate-EDTA).
To produce the gel and make wells where samples are loaded, use a casting tray and comb.
The device that contains the gel and buffer and creates the environment in which the electric field is applied is known as the electrophoresis chamber.
3. Power Supply
Produces the electric field required for the mobility of molecules.
Samples are combined with loading dye to see loading and track progress.
4. DNA ladder
A combination of known-size DNA fragments used as a reference to gauge the size of sample fragments is called a DNA ladder.
5. Staining agents
Substances that attach to nucleic acids and fluoresce when exposed to UV light to allow visibility, such as ethidium bromide or SYBR Safe.
Steps of Agarose Gel Electrophoresis
1. How to Prepare Gel
Heat the agarose powder until it dissolves in the electrophoresis buffer.Fill a casting tray with molten agarose and insert a comb to create wells.
2. Preparing the Sample
Combine loading dye with DNA or RNA samples to increase density and monitor the electrophoresis’s progress.
3. Filling the Gel
Put the gel into the electrophoresis chamber and pour the buffer over it.
Using a micropipette, carefully transfer the samples and DNA ladder into the wells.
4. Utilizing the Gel
Once the power source is connected to the chamber, operate the gel at a steady voltage (typically between 80 and 120V).
Track the dye front’s movement to determine when to end the run.
5. Visualization and Staining
If the gel hasn’t been previously dyed, stain it with a DNA-binding dye after electrophoresis.
Use a blue light transilluminator or UV light to see the DNA bands.
Applications
. Determining the size of DNA fragments following digestion by restriction enzymes is known as DNA fragment analysis.
.Verifying the size and amount of amplified DNA fragments by PCR product analysis.
.Verifying the size and integrity of RNA molecules is known as RNA analysis.
.Genotyping is the process of locating genetic variants like SNPs or microsatellites.
Cloning and sequencing: separating particular DNA segments for additional work.
Purification: Taking DNA fragments out of the gel so they may be sequenced or cloned.
Advantages and Limitations
Advantages:
Easy and economical to use.
able to resolve fragment sizes ranging widely.
harmless chemicals (well, with the exception of certain stains like ethidium bromide).
Limitations:
Conclusion
restricted resolution for parts that are extremely tiny or huge.
more time-consuming than electrophoresis in capillaries.
Possibility of UV radiation causing DNA damage.
An essential method in genetics, biochemistry, and molecular biology research is agarose gel electrophoresis. With the use of this technology, nucleic acids and proteins may be separated and analyzed according to their size and charge, yielding important information about their structure and function.
PCR, DNA sequencing, forensic investigation, and medical diagnosis are just a few of the many uses for the adaptable technology known as agarose gel electrophoresis. It is an easy-to-use, reasonably priced method that has grown to be indispensable in genetics and molecular biology research.
Frequently Asked Question
What is the principle of agarose gel electrophoresis application?
Agarose gel electrophoresis is used to resolve DNA fragments on the basis of their molecular weight. Smaller fragments migrate faster than larger ones; the distance migrated on the gel varies inversely with the logarithm of the molecular weight.
What are the 5 major steps used in electrophoresis?
There are several basic steps to performing gel electrophoresis that will be described below; 1) Pouring the gel, 2) Preparing your samples, 3) Loading the gel, 4) Running the gel (exposing it to an electric field) and 5) Staining the gel
What is agarose used in gel electrophoresis?
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar.
Related Article