How To Read & Interpret Gel Electrophoresis

Gel electrophoresis is an essential molecular biology technique used in biotechnology labs to separate and analyze nucleic acids (DNA fragments, RNA, and plasmids) and proteins based on their molecular weight.

The two types of widely used gel electrophoresis techniques include agarose gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both these electrophoresis techniques are further classified into native or denaturing gel electrophoresis.

In both methods, the biomolecules are forced by an electric field to move from one to the other end of the gel, which acts like a sieve. Let’s understand the separation of molecules through the gel matrix by taking an example of DNA separation.

During DNA gel electrophoresis, DNA samples are loaded to a negatively charged end of the gel (cathode). Thus, when an electric current is applied to the gel, the molecules move toward the positive electrode (anode) because of the negative charges carried by their sugar-phosphate backbone.

The rate at which DNA molecules move through the gel matrix is based on the size of the DNA. The small fragment of DNA will travel a greater distance than the larger DNA.

Separated DNA fragments are visible as bands at different locations within the gel matrix. These DNA bands help in determining the size of DNA molecules on an agarose gel alongside a DNA ladder (sample with known DNA fragment sizes).

Pulsed-field gel electrophoresis (PFGE) is another technique used to separate DNA fragments of more than 20-kilo base pairs (Kb) or chromosomes, which is not possible using traditional agarose gel electrophoresis. Here, the electrophoretic mobility of molecules depends on pulse time.

In this article, we will cover how you can interpret the gel electrophoresis results obtained after the experiment to draw accurate conclusions for your downstream applications.

Materials Needed for Gel Electrophoresis

Here’s the list of equipment and reagent you need to perform DNA gel electrophoresis assay in your labs:

Equipment

• Casting tray: An apparatus used to give shape to the gel and hold it during the workflow.
• Voltage source: Provides the electric current needed to perform the electrophoresis.
• Well combs: Used to create well for loading samples.
• Gel box: A container having attached electrodes and holds gel in a running buffer.
• Microwave: Used to dissolve agarose.
• UV light source: Used to visualize EtBr containing gels.

Figure: An illustration of equipment used for DNA electrophoresis.

Reagent

• TBE (Tris borate) buffer or TAE (Tris-acetate buffer): Also known as electrophoresis buffers. Both the buffers, TBE and TAE, contain varying concentrations of tris base and EDTA, with the former containing boric acid and the latter containing acetic acid as an additional component.

• Loading buffer: A high-density solution that facilitates the loading of DNA into the gel wells. It contains a colored dye and density agent. The colored dye offers visualization of separated DNA fragments and acts as a tracking dye that helps monitor the migration of DNA samples in the gel. They contain EDTA, Tris buffer, and either Cresol Red, Bromophenol Blue, Xylene cyanol FF, or Orange G as a loading dye.

• Ethidium Bromide (EtBr): One of the common reagents used in DNA electrophoresis to stain DNA for their visualization under UV lights. When using EtBr in gels, exercise caution and include a running buffer to counteract its positive charge, which causes it to migrate in the opposite direction of DNA.

• Agarose gel: A polysaccharide obtained from agar-bearing marine algae (sea kelp). It’s a polymer of alternating 3,6-anhydro-L-galactose and β-D-galactose units of agarobiose. It’s widely used as a gelling agent in labs for assays, such as immunodiffusion and nucleic acid gel electrophoresis techniques.

How To Read Gel Electrophoresis

Gel electrophoresis is a routine assay in molecular biology labs. It has a myriad of applications including:

• Determination of the size of DNA molecules after restriction digestion by restriction enzymes. For example, during restriction mapping of cloned DNA.
• Analyzing PCR products in genetic fingerprinting and genetic diagnosis
• Separation of restricted genomic DNA or RNA before Southern or Northern transfer

The results obtained in the technique are used for further downstream applications such as cloning, isoelectric focusing, bacterial transformation, Polymerase Chain Reaction (PCR), Genome or DNA sequencing, and blotting techniques.

Therefore, the correct interpretation of results obtained after the experiment is necessary to save the time and money invested in carrying out your research.

A small mistake, and you end up doing everything all over again.

That’s why here we’ve provided three steps that must be followed in order to accurately read gel electrophoresis and analyze the results.

DNA Ladder

A DNA ladder should be used and loaded in the first well of the gel. A ladder will have bands of known molecular weight that can be used to compare the samples that are going to be identified.

In gel electrophoresis, the loaded samples are separated in the form of bands. If using EtBr, which binds to DNA, the gel can be visualized using UV light. It reveals the intensity of bands and their location in the gel. High-quality buffers and chemicals need to be used in order to clear, sharp bands and good separation.

Figure: An illustration of the separation of DNA bands on a gel

Analyze Sample Changes

Gel electrophoresis is used to study and distinguish the target molecules from a sample containing different types of molecules. For example, while performing DNA or agarose gel electrophoresis, you need to determine cut plasmids, uncut plasmids, and nicked plasmids in a gel.

Ensure if there are any contaminants in the gel, such as in DNA gel electrophoresis, RNA contaminants appear above, and protein contaminants are seen below the DNA bands. In the case of PCR results, look for primer dimers to determine the accuracy of PCR amplification.

Interpret Results

The parameters to look for in a gel depend on the nature of your experiment. For example, for DNA fingerprinting you’d be comparing the DNA size of two samples, but while working with bacterial plasmids you might be interested in plasmid inserts.

To determine the size of the DNA in your sample, you can match its bands to the closest band in the DNA marker. Simply draw a horizontal line across from the marker bands and estimate the size of the DNA in your sample based on the closest match. To accurately determine the size of DNA fragments in base pairs, you need to calculate the relative mobility of each band.

This can be done by measuring the distance between the well and the tracking dye, and the distance between the well and the bands. Then, divide the sample band distance by the tracking dye band distance.

This gives relative mobility, which is used to plot a standard graph with ladder sizes to determine the size of the molecule in the study.

Figure: An illustration of Gel electrophoresis result and a graph showing DNA migration distance vs fragment size.

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Gel electrophoresis is a routine lab procedure performed in molecular biology labs to study and analyze biomolecules, such as DNA, RNA, and proteins. It’s generally followed by downstream applications, such as cloning, sequencing, and blotting experiments. Therefore, any mistake in performing the experiment or interpreting its results can lead to a great loss of money and time.

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