Agarose Gel Electrophoresis- Definition, Principle, Parts, Steps, Applications
Agarose gel electrophoresis is a widely used technique in molecular biology, biochemistry, genetics, and clinical chemistry. It is used to separate and analyze different types of molecules, such as DNA, RNA, and proteins, based on their size and charge.
Agarose is a natural polysaccharide extracted from seaweed. It forms a gel-like matrix when dissolved in a buffer solution and heated. The gel has pores of varying sizes that allow the movement of molecules under the influence of an electric field. The molecules are loaded into wells at one end of the gel and migrate towards the opposite end according to their size and charge. Smaller and more negatively charged molecules move faster and farther than larger and less negatively charged molecules.
Agarose gel electrophoresis can be used for various purposes, such as:
- Estimating the size of DNA or RNA fragments by comparing them with known standards
- Analyzing the products of polymerase chain reaction (PCR), such as in genetic testing or forensic science
- Separating DNA fragments after digestion with restriction enzymes, such as in cloning or mapping
- Separating RNA molecules before hybridization with probes, such as in northern blotting
- Separating proteins by their charge or molecular weight, such as in western blotting or immunoelectrophoresis
Agarose gel electrophoresis is a simple, inexpensive, and versatile method that can provide valuable information about the structure and function of biomolecules. It can also be combined with other techniques, such as staining, blotting, or sequencing, to enhance the detection and analysis of the molecules. In this article, we will discuss the principle, requirements, steps, applications, and advantages and disadvantages of agarose gel electrophoresis.
Agarose gel electrophoresis is based on the principle that charged molecules will migrate in an electric field towards the opposite charge. DNA molecules are negatively charged due to the phosphate groups in their backbone, so they will move towards the positive electrode (anode) when an electric current is applied. The agarose gel acts as a sieve that separates the DNA molecules according to their size and shape. Smaller and more compact molecules will move faster and farther than larger and more linear ones. The rate of migration also depends on other factors such as the concentration and composition of the agarose gel, the voltage and duration of electrophoresis, the ionic strength and pH of the buffer, and the presence of intercalating agents or other additives.
The separation of DNA molecules by agarose gel electrophoresis can be visualized by staining the gel with a fluorescent dye that binds to DNA, such as ethidium bromide. Ethidium bromide intercalates between the base pairs of DNA and emits orange-red light when exposed to ultraviolet (UV) light. The stained DNA bands can be observed and photographed under a UV transilluminator. The size of the DNA fragments can be estimated by comparing their migration distance with that of a DNA ladder, which is a mixture of DNA fragments of known sizes that are run alongside the samples. The DNA ladder serves as a reference or standard for determining the approximate size of the unknown DNA fragments.
To perform agarose gel electrophoresis, you will need the following equipment and supplies:
- An electrophoresis chamber and power supply. The chamber consists of a tank that holds the buffer solution and the gel, and two electrodes that generate an electric field across the gel. The power supply controls the voltage and current applied to the chamber. You can choose from different types and sizes of chambers and power supplies depending on your needs and preferences.
- Gel casting trays and combs. The trays are made of UV-transparent plastic and have open ends that can be sealed with tape while the gel is being cast. The combs are inserted into the trays to create wells in the gel where the samples will be loaded. The number and size of the wells depend on the comb design. You can also use different shapes and colors of combs to distinguish between different gels or experiments.
- Electrophoresis buffer. The buffer provides the ions that carry the electric current and maintain the pH of the system. The most common buffers used for agarose gel electrophoresis are Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE). TAE has a lower buffering capacity but is cheaper and less likely to interfere with downstream applications. TBE has a higher buffering capacity but is more expensive and may inhibit some enzymes or nucleic acid modifications.
- Agarose powder. Agarose is a natural polysaccharide extracted from seaweed that forms a gel when dissolved in buffer and cooled. Agarose gels have a porous structure that allows the separation of molecules based on their size and charge. The concentration of agarose in the gel determines the resolution and range of separation. Higher concentrations result in smaller pores and better resolution of smaller molecules, while lower concentrations result in larger pores and better separation of larger molecules.
- Loading buffer. The loading buffer is added to the samples before loading them into the wells. It has two main functions: (1) to increase the density of the samples so that they sink to the bottom of the wells, and (2) to provide tracking dyes that allow you to monitor the progress of electrophoresis. The loading buffer usually contains glycerol as a density agent, and bromophenol blue or xylene cyanol as tracking dyes. The tracking dyes have different molecular weights and colors, and migrate at different rates in the gel, depending on the agarose concentration and buffer used.
- Staining solution. The staining solution is used to visualize the nucleic acids in the gel after electrophoresis. The most common staining agent is ethidium bromide (EtBr), which intercalates between the base pairs of DNA or RNA and fluoresces under UV light. EtBr can be added to the gel or buffer before electrophoresis, or to the gel after electrophoresis. However, EtBr is also a potent mutagen and carcinogen, so it should be handled with care and disposed of properly. Alternatively, you can use safer staining agents such as SYBR Green, GelRed, or GelGreen, which have similar properties but lower toxicity.
- Transilluminator. The transilluminator is a device that emits UV light to excite the fluorescence of the stained nucleic acids in the gel. You can use a transilluminator with a built-in camera or a separate camera to capture images of your gel for documentation or analysis. You should wear protective goggles or glasses when using a transilluminator to avoid eye damage from UV exposure.
These are the basic requirements and instrumentation for agarose gel electrophoresis. Depending on your specific application, you may also need additional equipment or supplies such as molecular weight markers, restriction enzymes, DNA ladders, pipettes, tips, gloves, etc.
To perform agarose gel electrophoresis, you need to follow these steps:
- Prepare the gel. First, you need to dissolve agarose powder in an appropriate electrophoresis buffer (usually TAE or TBE) to make a gel solution. The concentration of agarose depends on the size of DNA fragments you want to separate. Typically, it ranges from 0.5% to 2%. You also need to add a fluorescent dye, such as ethidium bromide or SYBR Green, to the gel solution to stain the DNA. Then, you need to heat the gel solution in a microwave oven or a water bath until it boils and becomes clear. Next, you need to let the gel solution cool down slightly (to about 60°C) and pour it into a gel casting tray that has a comb inserted at one end. The comb will create wells for loading the samples. You need to wait until the gel solidifies at room temperature or in a refrigerator.
- Load the samples. After the gel is set, you need to remove the comb carefully and place the gel in an electrophoresis chamber that is filled with the same buffer as the gel. Then, you need to mix your DNA samples with a loading buffer that contains glycerol and a tracking dye. The loading buffer will make the samples sink into the wells and allow you to monitor their migration. You also need to load a DNA ladder or marker that contains DNA fragments of known sizes as a reference. You need to use a micropipette to transfer the samples and the ladder into the wells of the gel.
- Run the electrophoresis. Next, you need to connect the electrophoresis chamber to a power supply and apply an electric current. The current will make the negatively charged DNA molecules move towards the positive electrode (anode), which is usually red. The smaller DNA fragments will move faster and farther than the larger ones, creating a separation pattern on the gel. You need to run the electrophoresis until the tracking dye reaches the end of the gel or close to it. The duration of electrophoresis depends on the voltage, the size of the gel, and the concentration of agarose.
- Visualize and analyze the results. Finally, you need to turn off the power supply and disconnect the electrophoresis chamber. Then, you need to take out the gel and place it on a UV transilluminator or a blue light illuminator. The fluorescent dye in the gel will emit light when exposed to UV or blue light, making the DNA bands visible. You can take a picture of the gel using a camera or a gel documentation system. You can also cut out and purify specific DNA bands from the gel if needed. To analyze the results, you need to compare the positions and intensities of your sample bands with those of the ladder bands. You can estimate the sizes of your sample DNA fragments by plotting a standard curve using the ladder data. You can also quantify the amount of DNA in each band by measuring its fluorescence intensity and using a calibration curve.
The concentration of agarose gel is an important factor that affects the resolution of DNA fragments in agarose gel electrophoresis. The concentration of agarose is expressed as a percentage of the weight of agarose powder per volume of buffer solution (w/v). For example, a 1% agarose gel means that 1 gram of agarose powder is dissolved in 100 ml of buffer solution.
The concentration of agarose gel determines the size range of DNA fragments that can be separated effectively. In general, lower concentrations of agarose gel allow better separation of larger DNA fragments, while higher concentrations of agarose gel allow better separation of smaller DNA fragments. This is because lower concentrations of agarose gel create larger pores in the gel matrix, which allow larger DNA fragments to migrate faster. Conversely, higher concentrations of agarose gel create smaller pores in the gel matrix, which slow down the migration of larger DNA fragments and increase the resolution of smaller DNA fragments.
The table below shows some examples of the optimal concentration of agarose gel for different size ranges of DNA fragments:
|Size range of DNA fragments (bp)
|Optimal concentration of agarose gel (%)
The concentration of agarose gel can also affect the quality and clarity of the DNA bands in the gel. Higher concentrations of agarose gel tend to produce sharper and more defined bands, while lower concentrations of agarose gel tend to produce broader and more diffuse bands. However, higher concentrations of agarose gel also require longer electrophoresis time and more buffer consumption.
Therefore, the optimal concentration of agarose gel depends on the specific purpose and goal of the experiment. The concentration of agarose gel should be chosen based on the size range and resolution of the DNA fragments of interest, as well as the desired quality and clarity of the DNA bands in the gel. A trial-and-error approach may be necessary to find the best concentration for a particular experiment.
To help you understand the process of agarose gel electrophoresis better, here is a video animation that illustrates the steps involved and the principles behind it.
As you can see from the video, agarose gel electrophoresis involves the following steps:
- Preparing the agarose gel by dissolving agarose powder in a buffer solution and heating it until it melts. Then adding ethidium bromide, a fluorescent dye that binds to DNA, to the gel and pouring it into a casting tray with a comb that creates wells for loading samples.
- Loading the samples of DNA mixed with a loading buffer that contains glycerol and tracking dyes into the wells of the gel. The loading buffer helps the samples sink into the wells and also allows monitoring the progress of electrophoresis by the movement of the dyes.
- Applying an electric current across the gel using an electrophoresis chamber and a power supply. The electric field causes the negatively charged DNA molecules to migrate towards the positive electrode (anode) at different rates depending on their size and shape. Smaller and linear DNA molecules move faster than larger and circular ones.
- Visualizing the DNA bands on the gel using a UV transilluminator. The ethidium bromide intercalates between the base pairs of DNA and fluoresces under UV light, revealing the location and intensity of the DNA bands. A DNA ladder, which is a mixture of DNA fragments of known sizes, is run alongside the samples to provide a reference for estimating the size of the unknown fragments.
The video also explains some of the factors that affect the resolution and accuracy of agarose gel electrophoresis, such as:
- The concentration of agarose in the gel. A higher concentration of agarose creates smaller pores in the gel matrix, which can separate smaller DNA fragments better but also slow down their migration. A lower concentration of agarose creates larger pores in the gel matrix, which can separate larger DNA fragments better but also allow them to diffuse more.
- The voltage applied across the gel. A higher voltage can speed up the electrophoresis process but also generate more heat, which can damage or melt the gel. A lower voltage can reduce the heat but also prolong the electrophoresis time.
- The buffer used for preparing and running the gel. The buffer provides ions that carry the electric current and maintain a constant pH in the gel. The most common buffers used are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA), which have different buffering capacities and conductivity. TAE is cheaper and less conductive than TBE, but also less stable and more prone to pH changes. TBE is more expensive and more conductive than TAE, but also more stable and resistant to pH changes.
I hope this video animation has helped you understand agarose gel electrophoresis better. If you have any questions or comments, please feel free to leave them below. 😊
Agarose gel electrophoresis is a versatile and widely used method for separating and analyzing nucleic acids (DNA and RNA) and proteins. Some of the common applications of agarose gel electrophoresis are:
- Estimation of the size of DNA molecules: By comparing the migration of DNA fragments of unknown size with a standard DNA ladder of known sizes, one can estimate the approximate length of the unknown fragments. This is useful for determining the size of PCR products, plasmids, restriction fragments, etc.
- Analysis of PCR products: PCR (polymerase chain reaction) is a technique that amplifies a specific region of DNA using primers and a DNA polymerase enzyme. Agarose gel electrophoresis can be used to check the quality and quantity of the PCR products, as well as to confirm the presence or absence of specific amplification products. For example, in molecular genetic diagnosis or genetic fingerprinting, agarose gel electrophoresis can be used to detect mutations, polymorphisms, or variations in DNA sequences.
- Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to Northern analysis: Southern and Northern blotting are techniques that transfer nucleic acids from a gel to a membrane, and then hybridize them with a labeled probe that binds to a specific sequence. These techniques are used to detect and quantify specific DNA or RNA molecules in a complex mixture. Agarose gel electrophoresis is used to separate the restricted genomic DNA or RNA by size before transferring them to the membrane.
- The agarose gel electrophoresis is widely employed to estimate the size of DNA fragments after digesting with restriction enzymes, e.g. in restriction mapping of cloned DNA: Restriction enzymes are enzymes that cut DNA at specific sequences, generating fragments of different sizes. Restriction mapping is a technique that determines the location and order of restriction sites on a DNA molecule by comparing the sizes of the fragments produced by different restriction enzymes. Agarose gel electrophoresis is used to separate and visualize the restriction fragments.
- Agarose gel electrophoresis is commonly used to resolve circular DNA with different supercoiling topology, and to resolve fragments that differ due to DNA synthesis: Circular DNA molecules, such as plasmids or bacterial chromosomes, can exist in different forms depending on their degree of supercoiling (twisting) or nicking (breaking). Agarose gel electrophoresis can separate these forms based on their mobility in the gel. Similarly, agarose gel electrophoresis can also separate DNA fragments that differ in their synthesis mode, such as leading and lagging strands, Okazaki fragments, etc.
- In addition to providing an excellent medium for fragment size analyses, agarose gels allow purification of DNA fragments: Since purification of DNA fragments size separated in an agarose gel is necessary for a number molecular techniques such as cloning, it is vital to be able to purify fragments of interest from the gel. This can be done by various methods, such as cutting out the desired band from the gel and extracting the DNA using commercial kits or organic solvents, or by using electroelution or electroblotting techniques.
These are some of the main applications of agarose gel electrophoresis in molecular biology and biochemistry. Agarose gel electrophoresis is a simple, fast, and reliable method that can provide valuable information about nucleic acids and proteins.
Agarose gel electrophoresis is a widely used technique for separating and analyzing DNA, RNA, and proteins. It has many advantages and disadvantages that should be considered before choosing this method for a specific application.
Some of the advantages of agarose gel electrophoresis are:
- It is simple and easy to perform, requiring only a few basic equipment and reagents.
- It is versatile and can be used for different types of molecules, such as linear or circular DNA, single-stranded or double-stranded RNA, and various proteins.
- It is relatively inexpensive and can be performed with low-cost materials, such as agarose powder, buffer solution, and ethidium bromide dye.
- It is non-denaturing and does not alter the structure or function of the molecules being separated.
- It allows the recovery of the molecules from the gel after electrophoresis, which can be used for further analysis or manipulation.
Some of the disadvantages of agarose gel electrophoresis are:
- It has a limited resolution and cannot separate molecules that differ by less than 10% in size or charge.
- It is affected by factors such as gel concentration, buffer composition, voltage, temperature, and loading volume, which can influence the migration rate and pattern of the molecules.
- It can be time-consuming and labor-intensive, especially for large-scale or high-throughput applications.
- It can pose health and environmental hazards due to the use of toxic chemicals, such as ethidium bromide, which can cause DNA damage and mutations.
- It can generate heat and cause the gel to melt or the buffer to become exhausted during electrophoresis, which can affect the quality and accuracy of the results.
Therefore, agarose gel electrophoresis is a useful technique for separating and analyzing DNA, RNA, and proteins, but it also has some limitations and drawbacks that should be taken into account. Depending on the purpose and scope of the experiment, other techniques such as polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), or chromatography may be more suitable or complementary.
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