Getting Started with DNA Gel Electrophoresis: Your Guide and Introduction

by Gold Biotechnology, Inc.

DNA gel electrophoresis is one of the most widely used analytical techniques in molecular biology, providing a simple and reliable way to separate DNA fragments based on size. By applying an electric field to DNA molecules embedded in a porous gel matrix, researchers can tell whether DNA fragments of interest are present, intact, and approximately the expected size.

Despite the increasing availability of automated and high-throughput platforms, gel electrophoresis remains a routine experimental checkpoint ranging from PCR verification and cloning to restriction analysis and general DNA quality control.

DNA gel electrophoresis separates DNA fragments (which have a negative charge) by size as they migrate through an agarose gel under an electric field, allowing visualization and comparison of DNA fragment lengths.

This article provides a practical introduction to DNA gel electrophoresis. Rather than presenting a step-by-step protocol, the focus is on understanding how gels work, what information they can and cannot provide, and how to interpret results critically.

By emphasizing common assumptions, limitations, and sources of ambiguity, the goal is to help you use gel electrophoresis not just as a confirmation step, but as a meaningful diagnostic tool within molecular biology experiments.

Key Terms for DNA Gel Electrophoresis

Before diving into gel electrophoresis, it might help to know what various terms mean. You don’t have to memorize it, but you can use this section to scroll back to if you aren’t sure about what something means.

Agarose:

A gel-forming substance made from seaweed that is used to create the gel in DNA electrophoresis, providing a porous matrix that separates DNA fragments by size.

Artifacts:

Unexpected or misleading features on a gel, such as smears or distorted bands, that do not represent the true nature of the DNA samples but are caused by experimental issues.

Band:

A visible line on the gel that represents a group of DNA fragments of similar size that have migrated to the same position.

Contamination:

DNA species other than the desired product.

DNA Conformation:

The physical shape of a DNA molecule (such as linear, circular, or supercoiled), which affects how it moves through the gel.

DNA Fragment:

A piece of DNA of a specific length, often produced by cutting or copying DNA in the lab.

DNA Ladder:

A mixture of DNA fragments of known sizes used as a reference to estimate the size of DNA samples on a gel.

DNA Migration:

The movement of DNA through the gel toward the positive electrode when an electric current is applied.

Intercalating Dyes:

Fluorescent molecules that insert between DNA bases and enable DNA bands to be visualized.

Lane:

A vertical path in the gel where a single sample or ladder is loaded and separated.

Matrix (Gel Matrix):

The porous gel material (usually agarose) that DNA moves through, acting like a filter that separates fragments by size.

PCR (Polymerase Chain Reaction):

A laboratory method used to make many copies of a specific DNA fragment before running it on a gel.

Resolution:

How well a gel can separate DNA fragments that are similar in size.

Run Time:

The length of time the electric current is applied to the gel during electrophoresis.

Sample:

The DNA solution that is loaded into a well to be analyzed on the gel.

Well:

A small hole in the gel where DNA samples are placed before electrophoresis begins.

What is DNA gel electrophoresis?

DNA gel electrophoresis is a foundational technique used to separate and visualize DNA fragments based on size. By applying an electric field to DNA molecules embedded in a porous gel matrix (the agarose), you can assess fragment length, integrity, and relative abundance. Even in the presence of modern high-throughput technologies, gel electrophoresis remains a standard approach for assessing PCR amplification, restriction enzyme activity, cloning success, and DNA integrity.

Importantly, gel electrophoresis provides comparative and qualitative information rather than definitive molecular identity. It allows researchers to ask whether DNA fragments of the expected size are present, whether samples are intact or degraded, and whether reactions proceeded as intended, but not to determine the exact DNA sequence or fine structural features.

Core principle of DNA separation

The separation achieved during gel electrophoresis relies on two key properties of DNA: its uniform negative charge density and its movement through a porous matrix under an electric field. When voltage is applied, DNA migrates toward the positive electrode, with smaller fragments moving faster than larger ones.

If you imagine the agarose gel as a 3-dimensional mesh, something very small is going to get through the holes (pores) quicker than something larger. Therefore, it makes sense that smaller fragments will move through your agarose gel faster during a given run time than larger fragments.

Another real-world way to imagine this is how much easier and faster it would be to move a dollhouse chair to another room in your home than it would be to move a large couch. This is because the dollhouse chair is a lot smaller and can get through the obstacles of a normal home without trouble compared to large, human-sized pieces of furniture.

Agarose gels act as a molecular mesh because of its composition. Their pore size is determined primarily by agarose concentration, which in turn affects resolution across different DNA fragment size ranges.

Figure 2. Small DNA fragments migrate faster through the gel during a given run time. These smaller fragments are shown closer to the bottom of the gel whereas large DNA fragments migrate slowly and are closer to the top of the gel (nearer the wells).

Visualization and interpretation

DNA within the gel is visualized using fluorescent dyes that bind or associate with nucleic acids, such as ethidium bromide, GelGreen®, and GelRed®.

Intercalating dyes offer high sensitivity but may affect DNA structure or downstream usability, while non-intercalating dyes provide safer handling and compatibility with blue-light imaging systems.

Band position is commonly used to estimate fragment size by comparing it to the ladder, while band intensity is often treated as a proxy for DNA mass.

What this means is that on any given DNA gel, you’ll have a series of lanes. These lanes correspond to the well. In one well, you may have pipetted in your DNA ladder. In another, you may have pipetted in a sample, and in another you pipetted in another sample. Another well may contain your control.

Once your gel has completed its run, you can compare your lanes to the control, to each other, and to the ladder.

Your DNA ladder is made of a series of known DNA fragment sizes. It acts like a ruler within the gel. You can look at where in the lane your sample fragments are, compare them to where in the gel the ladder bands are and get a sense of the size of your fragment.

You can also look at how bright your sample showed up in the well to get a sense of abundance.

And you can compare your sample to others or to a control in order to make certain judgements about your results.

Let’s look at the example in figure 3, and see how we can use the ladder, and sample comparisons to make notes about the results.

DNA Gel Example with ladder and lane bands

Figure 3. An example of a gel with a ladder in lane 1. Different samples are in lanes 2-5. Lane 6 contains a negative control (water) where no band will show up.

Lane 1: Lane one in figure 3 is your DNA ladder. This is composed of DNA fragments of known sizes. This ladder in particular has a size range of 100 base pairs to 1,500 base pairs. Because the 100 base pair fragment is much smaller, it has traveled the furthest distance in the gel than the other fragments during the gel run time.

Lane 2: Lane 2 contains two fragments. When we compare them to the ladder (lane 1), we can estimate the two fragments to be 900 base pairs and 800 base pairs in size. Notice also, the band is thinner and a little faint. If you’ve loaded equal volumes for all samples then this indicates that the sample for lane 2 contains a lower concentration of DNA.

Lane 3: Lane 3 contains 3 fragments. Two of the fragments line up with lane 2. Though this does not prove it, it suggests that lane 3 contains the same DNA fragments as lane 2. You’ll need to assess if these are likely the same DNA fragments based on your experimental design. Or, you could confirm that these are the same DNA fragments by sequencing them. However, the third band at 1,500 base pairs suggests there is another DNA fragment within this sample.

Lane 4: Lane four also contains 2 fragments at the same size as lane 2. Therefore, it is possible the sample used for lane 4 contains the same DNA fragments as the sample in lane 2. Notice, however, that the bands are thicker and brighter relative to the sample in lane 2. If you loaded the same volume in lanes 2 and 4, this suggests the sample for lane 4 contains a higher concentration of DNA.

Lane 5: The sample used in lane 5 contains only one fragment. When compared to the ladder and the other samples, you will notice that it appears to be about the same size as the extra fragment in lane 3. This could mean (but would require more testing for confirmation), that the DNA sample that was in lane 5 is also present as the 3^rd larger band in lane 3.

Lane 6: Lane 6 is a negative control, where water was used instead of a DNA sample. This is why this lane is entirely blank. However, if a band showed up in this lane, and the same band showed up in all of your other lanes, this could signal contamination in your sample or setup.

When it comes to observed band intensity or brightness, fluorescence intensity is influenced by dye chemistry, fragment length, and imaging conditions, limiting quantitative accuracy. Gels are best used for relative comparisons within the same run rather than absolute measurements across experiments.

Gel electrophoresis answers limited questions and provides limited observations:

whether fragments of expected size are present
whether abnormalities are evident.
Having a DNA band of approximately the correct size is an encouraging sign, but it doesn’t guarantee that your DNA has the correct sequence or structure.

photo of a dna gel with bands - wikimedia commons

Figure 4. DNA bands visualized on an agarose gel with a blue light transilluminator (Ultrabem, 2023. CC0 1.0 Public Domain).

Sample preparation and loading

Sample quality directly affects the outcome of your gel. Residual salts, organic solvents, or detergents can alter DNA migration and cause smearing. Contamination can also ruin results.

Overloading DNA, particularly high-molecular-weight material, can obscure resolution and complicate interpretation.

DNA ladders are used as size markers to help estimate the length of DNA fragments. However, most ladders are made of straight (linear) DNA, so comparing them to circular DNA can be tricky.

Circular DNA can move through the gel at different speeds depending on its shape. For example, tightly coiled DNA usually moves faster than straight DNA of the same size, while relaxed or damaged DNA moves more slowly. Because of this, it is important to consider DNA shape when interpreting gel results. See this article for more details on interpreting DNA gel electrophoresis results.

Choosing the right gel and buffer during setup

Selecting appropriate gel and buffer conditions is critical for interpretable results. Agarose concentrations typically range from 0.5–2%, with lower concentrations being used to separate large fragments and higher concentrations being used to better separate smaller fragments.

When gels are too concentrated, it can reduce mobility and worsen band distortion. However, overly dilute gels compromise resolution.

The two most commonly used buffers are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). TAE offers faster migration and is often preferred for downstream DNA recovery, while TBE provides higher buffering capacity and sharper resolution for small fragments but can inhibit some enzymatic reactions if carried over. Buffer choice should reflect both resolution needs and downstream applications.

If you want to learn more about the differences between TAE and TBE buffers, and when to use each, we have a more detailed article here.

Running the gel: practical considerations

Electrophoresis conditions significantly influence band quality. Voltage is typically expressed as volts per centimeter (V/cm) between electrodes; higher voltages reduce run time but increase heat generation, which can distort bands and reduce resolution. Excessive heating often shows up as “smiling,” where bands curve upward at the edges of the gel.

Gel thickness and well geometry also matter. Thick gels retain heat and reduce resolution, while poorly formed wells can cause uneven loading and distorted migration.

Run time should be sufficient to separate fragments of interest without allowing smaller bands to migrate off the gel. Consistent buffer depth and even electrode contact help maintain uniform electric fields.

Common problems and troubleshooting

You’re not always going to get a perfect gel. In fact, people who have done this a thousand times still run into issues. So, let’s talk about some of the common issues that come up during gel electrophoresis.

· Smearing can indicate DNA degradation, overloading, or contamination with salts or solvents.

· Curved or “smiling” bands typically reflect uneven heating or voltage gradients.

· Poor resolution between similarly sized fragments may result from inappropriate gel concentration or excessive run speed.

Unexpected migration patterns often trace back to DNA conformation, buffer composition, sample impurities, or may illustrate issues with the upstream experiment.

Rather than treating gels as purely confirmatory, experienced users interpret artifacts as clues to upstream experimental issues.

For more about troubleshooting gel electrophoresis, we have a few resources:

· How to be a Better Troubleshooter in your Laboratory

· Troubleshooting DNA Ladders

· Getting No Bands in your PCR Results – Tips to Help

· Weak PCR Band Results or Smearing - What To Do

DNA gel electrophoresis is a robust, accessible method for analyzing DNA size, integrity, and relative abundance. Its strengths lie in simplicity, flexibility, and diagnostic value within molecular biology. However, it offers limited resolution, minimal quantitative precision, and no sequence-level information.

Used thoughtfully, gel electrophoresis provides rapid, reliable insight into experimental progress. When questions extend beyond size and presence, more specialized techniques are required, but the gel often remains the first place researchers look.

References

Ultrabem. (2023, March 17). Agarose gel electrophoresis of DNA [Image]. Wikimedia Commons. https://commons.wikimedia.org/wiki/File:Agarose_gel_electrophoresis_of_DNA.png

AI Disclosure

In order to write this article, AI assisted extensively in the research, planning and writing of this paper. However, the article was human-edited, with human re-writing and re-drafted multiple times. Images in this article (except for figure 4) were human-generated and belong to Gold Biotechnology.

Getting Started with DNA Gel Electrophoresis: Your Guide and Introduction

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