Agarose Gel Electrophoresis: History, Principle, and Procedure

Agarose Gel Electrophoresis is one of the most widely used laboratory technique to separate biomolecules namely DNA, RNA and proteins based on their size, length, weight, shape and charge. By comparing the DNA bands of unknown sample with the known and standard one (marker), we can estimate the length and size of the unknown DNA sample.

Table of Contents

1. History of Gel Electrophoresis
2. Principle of Gel Electrophoresis
3. Procedure of Gel Electrophoresis
4. Preparing the gel

History of Gel Electrophoresis

Gel Electrophoresis is one of the most powerful and useful technique for studying DNA in different branches of Life Sciences. This technique was invented and developed by several scientists during 20th century. The basic principle of electrophoresis is the movement of charged molecules in an electric field. During early stage of electrophoresis invention and development, the separation of molecules was achieved in liquids. But this liquid electrophoresis was incapable of separating DNA and proteins molecules. Hence, there was a need to improve the technique. In 1940, Dr. Oliver Smithies was the first to use starch in place of liquid in electrophoresis for which he had received Nobel Prize. The starch electrophoresis was able to separate protein molecules which was significant improvement in the field of electrophoresis. In 1960, Dr. David I. Brown replaced starch with agarose, making this technique easier and effective in separating the molecules.

Principle of Gel Electrophoresis

The principle of gel electrophoresis is the movement of charged biomolecules in an electric field. The gel matrix is cast in rectangular shape and it is composed of agarose or polyacrylamide. The gel is permeable matrix acts like a sieve. An electric current is applied across the gel, under the influence of electric current, the negatively charged molecules migrate or run towards positive electrode – anode whereas, the positively charged biomolecules run towards negative electrode – cathode. The movement of charged ions is called as migration. As DNA and RNA are negatively charged molecule, they migrates towards anode. The charge of the protein depends upon the isoelectric point of respective protein and pH of the buffer system.

The migration rate or speed of these charged biomolecule is influenced by their size and weight. The lighter and shorter biomolecule runs faster than the heavier and longer ones. Hence, shorter DNA strands will run more quickly than longer DNA strands. Due to their difference in charge and size, the biomolecules get separated and appears like distinct bands on the gel. We can visualise them in the form of bands because of the use of dyes, radio-labelled and florescent tags. Hence, agarose gel electrophoresis is used to conduct quantitative studies by comparing the position of bands of unknown sample with the known standards. It is widely used to DNA fingerprinting and characterisation of biomolecules.

Procedure of Agarose Gel Electrophoresis –

1. Gel Preparation – The agarose or polyacrylamide powder is mixed with appropriate quantity of buffer to obtain gel. The most preferable buffers are TBE (Tris-Borate-EDTA) or TAE (Tris-Acetate-EDTA). To obtain gel, the mixture of agarose or polyacrylamide and buffer is heated and poured into a mold. A comb is inserted into a casting gel to create wells where the samples can be loaded. As the mixtures cools down, it solidifies and forms a gel and the comb is removed.
2. Preparation of Electrophoresis Chamber – It houses the gel and running buffer. The chamber consist of positive and negative terminal at opposite ends.
3. Sample Preparation – The isolated DNA, RNA or protein sample are mixed with loading dye. The loading dye consist of tracking dye and density agent. The tracking dye are chromogenic dye ( Eg – Xylene cyanol FF, Orange G, Bromophenol Blue, or Cresol Red) which can physically associate with DNA, RNA or proteins allowing the visualise their movement. Density agent like Ficoll, sucroseor glycerol also get physically associated with biomolecules without disturbing the integrity of biomolecules. The purpose of density agent is to sink the biomolecules into the wells while loading the sample. Hence, loading dye is not only used to visualise the migration of biomolecules but for also the sinking of biomolecules into the well while loading the sample.
4. Loading the gel – Once the gel is solidified and the comb is removed, the prepared sample is loaded into the wells using micropipette. The standard DNA ladder or molecular weight marker is loaded for reference.
5. Electrophoresis – The electrophoretic equipment consist of two chambers separated by buffer filled chamber where the gel is submerged. The two positive and negative electrodes are placed at the opposite ends of those two chambers. The gel is submerged in appropriate buffer solution and the electric current is supplied. Under the electric current, the positive ions moves towards anode and the negative ions moves towards cathode. The rate of migration depends upon size and weight of the molecules.
6. Run the gel – The gel is allowed to run for atleast 30 min to several hours depending upon desired results. Due to the difference of size and weight the molecules appears in the form of bands on the gel.
7. Visualisation – To visualise DNA, the gel is stained with ethidium bromide dye. The ethidium bromide dye gets intercalate with DNA making it visible under UV light. The visualisation and documentation of DNA is done by Gel Doc system.
8. Analysis- The separated band of unknown samples are compared with the molecular marker. Comparing with molecular marker allows us to estimate the possible molecular weight of unknown DNA sample.

Preparing the gel

• Agarose gels are typically used to visualise fragments of DNA. The concentration of agarose used to make the gel depends on the size of the DNA fragments you are working with.
• The higher the agarose concentration, the denser the matrix and vice versa. Smaller fragments of DNA are separated on higher concentrations of agarose whilst larger molecules require a lower concentration of agarose.
• To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted.
• The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make wells for the sample to be pipetted into.
• Once the gel has cooled and solidified (it will now be opaque rather than clear) the comb is removed.
• Many people now use pre-made gels.
• The gel is then placed into an electrophoresis tank and electrophoresis buffer is poured into the tank until the surface of the gel is covered. The buffer conducts the electric current. The type of buffer used depends on the approximate size of the DNA fragments in the sample.

Key Considerations and Troubleshooting

• Gel concentration: Choose the appropriate gel concentration based on the size of the molecules being separated.
• Buffer selection: Select a suitable buffer that provides optimal conductivity and maintains pH during electrophoresis.
• Voltage and current: Adjust the voltage and current to avoid excessive heating and gel melting.
• Sample loading: Load the samples carefully to avoid overloading the wells, which can lead to poor resolution.
• Visualization: Choose a suitable staining method that provides adequate sensitivity and specificity.

Conclusion

Agarose gel electrophoresis is a cornerstone technique in molecular biology, providing a simple yet powerful method for separating and analyzing biomolecules. Its versatility and ease of use have made it an indispensable tool in research laboratories worldwide. As technology continues to advance, new variations of this technique, such as pulsed-field gel electrophoresis, are being developed to address more complex challenges in molecular biology.