Five Main Nucleic Acid Gel Electrophoresis Steps

The five main steps in nucleic acid gel electrophoresis are gel preparation, sample and ladder preparation, electrophoretic run, sample visualization, and gel documentation. Click on the tiles to deep dive into each step.

Gel preparation

Sample and ladder preparation

Electrophoretic run

Sample visualization

Gel documentation

Agarose and acrylamide are the two most common gel matrices utilized in the electrophoretic separation of nucleic acids. Both materials are nonreactive with the nucleic acids, and they form three-dimensional matrices with pores, which act as a sieve, for separation of nucleic acid. To efficiently resolve nucleic acids fragments of different sizes, the pore sizes are adjusted by varying the concentration of agarose or acrylamide in the gel matrix. The key differences between the two methods are listed in Table 1.

The choice between agarose gels and polyacrylamide gels depends on:

• Size range
• Desired resolution of nucleic acid samples
  • Agarose forms matrices with pore sizes ideal for separating nucleic acid molecules in the range of 0.1–25 kb. Polyacrylamide, on the other hand, forms smaller pore sizes, which resolve nucleic acid molecules smaller than 1 kb. In some cases, single-base resolution between fragments of <100 bp may be obtained with polyacrylamide gels [1].
• Preferred gel casting and sample recovery methods.

When an agarose solution is heated and cooled, it forms a gel matrix with pore sizes ranging from 50 to 200 nm in diameter, as governed by gel concentration. At a temperature above 90°C, agarose melts and forms random coils. Upon cooling, two agarose chains form helical fibers linked by hydrogen bonds. Further cooling below the gelling point (usually <40°C) results in networks of helical bundles held together by more hydrogen bonds, forming a gel with three-dimensional meshes (Figure 2) [2,3]. Because of the hydrogen bonds, gel formation of agarose is reversible by heat. This property of agarose aids in extraction of nucleic acids by melting gel slices containing fragments of interest.

The agarose used in gel electrophoresis may be standard agarose or low melting point (LMP) agarose.

LMP agarose melts around 65°C (for a 1% gel), a relatively low temperature, compared to the melting point of standard agarose that ranges between 90°C and 95°C.

The advantages of LMP agarose, include:

• Enables gentle extraction of large nucleic acid fragments (>10 kb) from gels, allowing nucleic acid fragments remain intact
• LMP agarose’s low gelling temperature (~25°C) also makes it ideal for in-gel enzymatic reactions, such as ligation, where enzymes are active within a semi-solid agarose solution

Agarose for gel preparation is commonly available in powder and tablet format. To streamline the workflow and save time, precast agarose gels are run on commercially available systems. These integrated systems have gel running, gel visualization, and band analysis.

Protocol for agarose gel preparation for electrophoresis

Polyacrylamide is a polymer of acrylamide monomers crosslinked with bis-acrylamide, or N,N′-methylene-bis-acrylamide. The crosslinker, bis-acrylamide, contains two units of acrylamide joined by a methylene bridge. Their polymerization is by free radical reactions—usually initiated by ammonium persulfate (APS), which is catalyzed by TEMED (N,N,N′,N′-tetramethylethylenediamine) (Figure 4). The concentration of APS and TEMED determine the rate of polymerization at a given temperature.

Components of polyacrylamide gels are acrylamide and a crosslinking agent, bisacrylamide, also called “bis”; both ingredients are available as powder, but pre-made solutions are commonly used for convenience. The powder and liquid forms are known neurotoxins and must be handled with care using protective labwear. The total concentration of acrylamide plus bisacrylamide (expressed as %T) in a gel determines the pore size, which ranges between 20 and 150 nm in diameter [5]. The higher the percentage, the smaller the pore size. The pore size and the size of molecules resolved are proportional. Commonly used gel percentages are listed in Tables 4 and 5 [4].

The relative composition of %T, the weight percentage of bisacrylamide (crosslinker) to total acrylamide (%C) is critical to the pore size of polyacrylamide gels and sample separation (Table 6) [5].

%T and %C can be expressed as:
%T (w/v) = [grams of (acrylamide + bisacrylamide) / milliliters of buffer] x 100%
%C (w/w) = [grams of bisacrylamide / grams of (acrylamide + bisacrylamide)] x 100%

Table 6. Standard polyacrylamide gel composition

Agarose and polyacrylamide gels are prepared using an ionic solution with electrical conductivity to enable nucleic acid mobility during electrophoresis. The same buffer type is usually used for both the gel and the running buffer during an electrophoretic run to maintain the same pH and ionic strength. The two most common buffers for nucleic acid electrophoresis are Tris-acetate with EDTA (TAE) and Tris-borate with EDTA (TBE), both with pH close to neutral to favor negative charges on the nucleic acids.

When preparing gels for electrophoresis, it is important to choose the appropriate buffer solution to help ensure proper migration of nucleic acids. Agarose and polyacrylamide gels are typically prepared using an ionic solution with electrical conductivity, and the same buffer type is usually used for both the gel and the running buffer to maintain a consistent pH and ionic strength. Two of the commonly used buffers for nucleic acid electrophoresis are Tris-acetate with EDTA (TAE) and Tris-borate with EDTA (TBE) [6,7], both of which have a pH close to neutral to favor negative charges on the nucleic acids.

For the analysis of single-stranded DNA or RNA, agarose and polyacrylamide gels are often prepared and run under denaturing conditions. Denaturing conditions disrupt hydrogen bonds between nucleic acids, reducing the formation of secondary structures such as hairpin loops. Denaturing electrophoresis is therefore more routine for RNA separation and analysis. Common denaturing buffers used in nucleic acid electrophoresis include:

• For agarose: glyoxal and DMSO in sodium phosphate buffer, NaOH-EDTA buffer, and formaldehyde or formamide in MOPS buffer
• For polyacrylamide: urea in TBE buffer

Learn more about buffer selection in gel runs

When running a gel, it is important to include a reference sample containing nucleic acids of known sizes, called a standard or marker or ladder, for size estimation of the samples of interest. Factors to consider when choosing an appropriate ladder for a given sample include:

• Ladder type: DNA or RNA
• Fragment structure: single-stranded or double-stranded 
• Conformation: supercoiled, open circular, or linear to ensure appropriate comparisons of migration 
• Number of DNA/RNA fragments and their separation patterns for proper size estimation
• Intended uses such as whether the ladder is designed for qualitative analyses or accurate quantitative measurements
• Suitability of the DNA/RNA ladder for the type of gel used; for example, precast gels recommend specific ladders designed for optimal runs
• Nature of loading dyes, to avoid obscuring the bands of interest (Figure 5)
• Compatibility of loading buffer with the gel used (e.g., salt concentration of the buffer may impact migration of the sample)
• Ladders from different manufacturers with the same description (e.g., 1 kb or 100 bp) may vary in the number, size, and intensity of DNA fragments (Figure 6), so refer to manufacturer’s guide before use for information on ladder composition

Learn more on the effects of structure and conformation on sample mobility

RNA ladders are usually provided with a loading buffer containing a denaturant. Denaturants help maintain RNA in single-stranded form, allowing more predictable sample migration and separation results. When performing RNA gel electrophoresis, DNA ladders should be avoided, because their use under denaturing conditions can lead to atypical separation patterns due to separation of the double strands.

For visualization and detection, the DNA bands must be well separated. For proper separation of the bands of interest, the volume of DNA to be loaded into each well must be calculated. For detection with a fluorescent dye, the recommended concentration is 1–100 ng/band of DNA; the minimal detectable quantity depends on the stain used. Overloading a sample or ladder can result in smearing of bands and masking those nearby, resulting in poor resolution, particularly when the fragments are of similar sizes (Figure 7A).

In preparation for gel electrophoresis, gel loading buffers typically made as 6X or 10X stock solutions are added to samples (and ladders, when needed). Components of loading buffers include the following:

• A density ingredient, such as glycerol or sucrose, increases viscosity of the samples, helping ensure that the samples sink into the wells.
• Salts, such as Tris-HCl, create environments with favorable ionic strength and pH for the samples. Loading buffers with high salt concentrations may produce broader or distorted bands and smears.
• A metal chelator, such as EDTA, prevents nucleases present in the sample from degrading nucleic acids.
• Dyes provides color for easy monitoring of sample loading, progress of the electrophoretic run, and pH changes. Some loading buffers may contain more than one dye, to track migration of molecules of varying sizes in a sample more efficiently.

Additives in loading buffers
The loading buffer may include detergents or reducing agents, such as SDS, urea, and formamide, for denaturing. These additives can disrupt molecular interactions between and within nucleic acid molecules, promoting linearity or single-stranded conformation of the molecules. Samples should be heated with denaturants in loading dye to obtain optimal separation results (Figure 9A). For the electrophoresis of double-stranded DNA from enzymatic reactions, SDS may be added to the loading buffer to disrupt interactions between proteins and nucleic acids, can prevent alteration of sample mobility (Figure 9B).

After preparation of gels, standards, and samples electrophoresis is performed. The gel must be completely solidified before removal of the comb and addition of the running buffer. The gel comb should be lifted upward, smoothly, and steadily, to avoid tearing the gel and distorting the wells. After buffer addition and comb removal, care should be taken to remove air bubbles that may be trapped in the wells. For polyacrylamide gels, the wells should be thoroughly rinsed with buffer to remove residual unpolymerized acrylamide.

A running buffer, which is an ionic solution with buffering capacity, is routinely used in gel runs. Running buffers allow current flow while impeding potential pH changes. During electrophoresis, the negative electrode becomes more basic and the positive electrode more acidic because of electron flow; this results in electrolysis of water and shifts in pH (Table 10). Release of hydrogen and oxygen gases causes bubbling from the electrodes, a telltale sign of a running gel. Ideally, the running buffer and the gel preparation buffer should be the same to help ensure efficient conductivity.

Table 10. Chemical reactions and pH changes at the two electrodes.

The choice of buffer for electrophoresis depends on sample sizes, run time, and post-electrophoresis processes, with Tris-acetate EDTA (TAE) and Tris-borate EDTA (TBE) being the two most common buffers (Table 11) [2,9]

• For separation of lower molecular weight samples, such as DNA <1,000 bp, TBE buffer is more advantageous 
• For denaturing electrophoresis, which is used to resolve molecules that tend to form secondary structures, such as RNA, TBE buffer is usually used; for example, polyacrylamide gels are primarily prepared with TBE buffer supplemented with 7–8 M urea or a similar denaturant to maintain single-strandedness of the nucleic acids
• For separation of nucleic acids of larger molecular weights, such as DNA of ≥12–15 kb, TAE buffer, together with low field strength (1–2 V/cm), is preferred; TAE buffer promotes larger apparent pore sizes of the gel, reduces electroendosmosis, and lowers field strength, all of which help decrease the tendency of large molecules to smear [6]

Table 11. Buffer selection guide

Gels should be completely submerged in buffer to allow ion flow and prevent gel drying (Figure 11A). However, when running a horizontal gel like an agarose gel, the depth of buffer over the gel should be no higher than 3–5 mm. Excess buffer over the gel (>5 mm) may result in decreased nucleic acid mobility, increased band distortion, and overheating. 

The running buffer of denaturing agarose gels may be made in different buffers, such as sodium phosphate and MOPS. It is important to select a running buffer that is compatible with the gel used.

To start a gel run, an electrical potential is applied across the gel with constant voltage, current, or power Constant voltage is commonly employed in nucleic acid electrophoresis, with voltage typically set at 5–10 V/cm.

Voltage to be applied (V) = distance between the electrodes (cm) x recommended V/cm

Additional information on the impacts of voltage on electrophoretic runs

Table 12. Run conditions based on fragment size

In some cases, a temperature probe may be connected to the gel apparatus to help control cooling and heating of the buffer. For electrophoretic runs less than two hours, cooling and recirculating the buffer may improve separation of the samples. For denaturing gels, allowing the buffer to heat up to 55°C may improve results by enhancing single-stranded forms of nucleic acids.

After a gel run is complete, the samples must be visualized. Since nucleic acids are not visible under ambient light, special detection methods are required for visualization. In the tabbed section below, Tables 13, 14, and 15 list the various methods that offer differing ranges of sensitivity and benefits in sample detection.

Mode of action of stains
Fluorescent dyes are most widely utilized in nucleic acid detection due to their ease of use and high sensitivity. The dye molecules bind or intercalate with DNA molecules at specific sites (Figure 14). When excited with an appropriate wavelength, the dye fluoresces or emits visible light. The intensity of fluorescence correlates to the amount of nucleic acid bound, which is the basis for detection and quantitation of nucleic acids in electrophoresis. Ethidium bromide and SYBR stains for nucleic acids are the most widely used fluorescent stains.

Guidelines on nucleic acid stain selection 

Two common approaches to staining nucleic acid samples are:

1. In-gel, where stain is incorporated into the gel (and running buffer)
2. Post-electrophoresis, where the gel is stained in a separate bath after the run is complete

The advantages and challenges of the two methods are listed in Table 16.

Table 16. Benefits and considerations of in-gel vs post-electrophoresis staining

In-gel staining is more convenient and requires less dye for visualization. However, the positively charged stain migrates in the direction opposite of nucleic acids, which can impact detection of samples of lower molecular weight, especially during a long run (Figure 15A). In addition, dyes binding to nucleic acids may alter the sample’s migration, a phenomenon known as gel shift, where samples do not run true to size (Figure 15B). Furthermore, high levels of intercalating dyes included in gels may change the conformation of supercoiled plasmid DNA, altering their mobility in electrophoresis [10].

Since post-electrophoresis staining does not affect samples during electrophoresis, it is the preferred method for accurate sizing of samples. However, the method adds time to the workflow, uses more dye, generates more hazardous waste when using dyes like ethidium bromide, and requires destaining steps prior to visualization.

Introduction of less toxic alternatives such as SYBR Safe DNA gel stain is tilting the preferences towards in-gel staining.

After visualization, nucleic acid gels are documented for record and analysis of electrophoresis results. If samples are stained with a fluorescent dye, special equipment is required to excite the dye with an appropriate light source to both visualize and capture a gel image. The two common light sources used are the epi-illuminator and the transilluminator (Table 17,Figure 17).

Table 17. Main differences between using an epi-illuminator or transilluminator for visualizing gel electrophoresis results

For electrophoresis of radiolabeled nucleic acids, the gel is exposed to X-ray film after electrophoresis for documentation, a process called autoradiography. The intensities of radiolabeled bands may be measured by densitometry for quantitation. 

Invitrogen E-Gel Electrophoresis Systems integrate gel running platform and image capture device in a single instrument. These benchtop instruments have a small footprint and combine the convenience of quick real-time nucleic acid analysis and high-quality visualization. The E-Gel electrophoresis systems are available in both high- and standard-throughput capabilities. Additionally, these systems operate with precast gels, which eliminates several steps in the traditional workflow.

In conclusion, nucleic acid electrophoresis workflows have multiple steps and reagents to separate and analyze samples. Choosing the right reagents to suit samples and optimizing the workflow can help improve the results of electrophoresis. Optimizing electrophoresis thus yields quality nucleic acid samples for downstream applications.

1. Stellwagen NC (1998) DNA Gel Electrophoresis. In: Tietz D (editor), Nucleic Acid Electrophoresis (Springer Lab Manual). Heidelberg: Springer. pp 1–53.
2. Yilmaz M, Ozic C, Gok I (2012) Principles of Nucleic Acid Separation by Agarose Gel Electrophoresis. In: Magdeldin S (editor), Gel Electrophoresis: Principles and Basics. Rijeka: InTech. pp 33–40.
3. Lee SV, Bahaman AR (2012) Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis. In: Magdeldin S (editor), Gel Electrophoresis: Principles and Basics. Rijeka: InTech. pp 41–56.
4. Green MR, Sambrook J (2012) Analysis of DNA. In: Molecular Cloning: A Laboratory Manual (4th ed). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. pp 81–156.
5. Barril P, Nates Silivia (2012) Introduction to Agarose and Polyacrylamide Gel Electrophoresis Matrices with Respect to their Detection Sensitivities. In: Magdeldin S (editor), Gel Electrophoresis: Principles and Basics. Rijeka: InTech. pp 3–14.
6. Brody JR, Kern SE (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem 333(1):1–13.
7. Stellwagen NC (1998) Apparent pore size of polyacrylamide gels: comparison of gels cast and run in Tris-acetate-EDTA and Tris-borate-EDTA buffers. Electrophoresis 19(10):1,542–1,547.
8. Thermo Fisher Scientific Inc (2010) Nucleic Acid Detection and Analysis. In: Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies. pp 349–360.
9. Hassur SM, Whitlock HW Jr (1974) UV shadowing--a new and convenient method for the location of ultraviolet-absorbing species in polyacrylamide gels. Anal Biochem 59(1):162–164.
10. Sigmon J, Larcom LL (1996) The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis. Electrophoresis 17(10):1,524–1,527.