Loading sample into agarose gel electrophoresis

Gel electrophoresis is a common laboratory technique to isolate nucleic acids and proteins. Nucleic acid electrophoresis uses a gel matrix to separate DNA and RNA fragments based on size and molecular weight. Gel electrophoresis is vital in all molecular biology workflows. Gel electrophoresis involves a series of steps to separate and analyze nucleic acid samples.

Five key steps in nucleic acid gel electrophoresis

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.

1. Selection and gel preparation for electrophoresis

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.

 

Table 1. Polyacrylamide gel vs agarose gel

 AgarosePolyacrylamide
SourcePolysaccharide polymer from red algaeSynthetic
Gel formationDissolves in water and forms extensive hydrogen bondsPolymerizes in the presence of crosslinking agent bis-acrylamide
Gel castingMelt and solidifyInitiate chemical reactions
Nucleic acid recoveryMelt and extractDissolve, and diffuse or electroelute
DNA separation range50–50,000 bp5–3,000 bp
Resolving power5–10 nucleotidesSingle nucleotide

What is agarose?

Agarose is a purified form of agar, a carbohydrate which forms the structural component of the cell walls of marine red algae. Agarose is a linear polymer with a molecular weight of ~120,000, comprising 800–1,000 monosaccharides. Agarose chains consist of a repeating heterodisaccharides—D-galactose and 3,6-anhydro-α-L-galactose linked by a β-1,4 glycosidic bond. The disaccharide unit, also known as agarobiose, forms a chain connected by a-1,3 linkages (Figure 1).

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.

Figure 2 . Effect of temperature on agarose structure in solution. At high temperatures, agarose forms random coil structures. As agarose cools in solution, they form helical fibers then tight helical bundles.

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

Preparing agarose gels

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

Recommended percentage of agarose gels for separation of DNA fragments

Gel percentage controls pore size, and thereby influences the separation and resolution of nucleic acid fragments. In general, higher-percentage gels result in better separation and resolution of smaller fragments (Figure 3). However, high-percentage gels may be turbid and interfere with visualization. Despite improved visibility, low-percentage gels can be fragile and difficult to handle. Tables 2 and 3 provide recommended agarose gel percentages for the separation of DNA fragments of different lengths [4].

To make your own agarose gels, the gel % is calculated as:

Gel % (w/v) = (g of agarose / mL of buffer) x 100%

Figure 3. Mobility of DNA fragments in agarose gels of different percentages. The same DNA ladder was separated on 1%, 2%, and 3% agarose gels under the same conditions, including run time. The 1 kb fragment is indicated by red asterisks for comparison.

Table 2. Range of efficient separation in low percentage gels (0.5–1.5%)

Gel percentageRange of efficient separation (bp)
0.52,000–50,000
0.61,000–20,000
0.7800–12,000
0.8800–10,000
0.9600–10,000
1.0400–8,000
1.2300–7,000
1.5200–3,000

Table 3. Range of efficient separation in high percentage gels (2–5%)

Gel percentageRange of efficient separation (bp)
2.0100–2,000
3.025–1,000
4.010–500
5.010–300

Table 2. Range of efficient separation in low percentage gels (0.5–1.5%)

Gel percentageRange of efficient separation (bp)
0.52,000–50,000
0.61,000–20,000
0.7800–12,000
0.8800–10,000
0.9600–10,000
1.0400–8,000
1.2300–7,000
1.5200–3,000

Table 3. Range of efficient separation in high percentage gels (2–5%)

Gel percentageRange of efficient separation (bp)
2.0100–2,000
3.025–1,000
4.010–500
5.010–300

What is a polyacrylamide gel?

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.

Preparing polyacrylamide gels

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].

Table 4. Recommended percentages of denaturing polyacrylamide gels for separation of nucleic acid fragments

Denaturing gels are used to resolve single-stranded nucleic acids in their linear form, so sizes are indicated in bases.

Polyacrylamide gel (with bis, at 19:1), %Range of efficient separation
4.0100–500 bases
5.070–400 bases
6.040–300 bases
8.030–200 bases
10.020–100 bases
15.010–50 bases
20.05–30 bases
30.01–10 bases

Table 5. Recommended percentages of non-denaturing polyacrylamide gels for separation of nucleic acid fragments

Non-denaturing gels are mainly used with double-stranded nucleic acids (bp: base pairs).

Polyacrylamide gel (with bis, at 19:1), %Range of efficient separation (bp)
3.5100–1,000
5.080–500
8.060–400
12.050–200
15.025–150
20.05–100

Table 4. Recommended percentages of denaturing polyacrylamide gels for separation of nucleic acid fragments

Denaturing gels are used to resolve single-stranded nucleic acids in their linear form, so sizes are indicated in bases.

Polyacrylamide gel (with bis, at 19:1), %Range of efficient separation
4.0100–500 bases
5.070–400 bases
6.040–300 bases
8.030–200 bases
10.020–100 bases
15.010–50 bases
20.05–30 bases
30.01–10 bases

Table 5. Recommended percentages of non-denaturing polyacrylamide gels for separation of nucleic acid fragments

Non-denaturing gels are mainly used with double-stranded nucleic acids (bp: base pairs).

Polyacrylamide gel (with bis, at 19:1), %Range of efficient separation (bp)
3.5100–1,000
5.080–500
8.060–400
12.050–200
15.025–150
20.05–100

Composition of bisacrylamide

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

Acrylamide:bis%CRelative pore sizesApplications
19:15%SmallDNA and denaturing gels
29:13.3%MediumssDNA and RNA in nondenaturing gels
37.5:12.7%LargeProtein gels

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.

Buffer choice in gel preparation for electrophoresis

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

2. Preparing standards and samples

Nucleic acid ladder selection guidelines

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.

Sample and standard preparation

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).

Samples and ladders are prepared in a loading dye in a buffer such that the final volume will typically occupy at least 30% of the well volume. Using a smaller volume may result in band distortion, due to poor distribution in the well (Figure 7B). For samples containing DNA-binding proteins or cohesive ends, the mixture may need to be heated in a loading dye with SDS prior to gel loading, because protein binding and interaction between the DNA fragments may cause poor separation (Figure 7B).

Loading dye and buffer choice

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.

Typically, loading dyes are small and negatively charged molecules so that they migrate in the same direction as the nucleic acids. Some display pH-dependent colors, serving as pH indicators for samples during loading and running (Figure 8A). Commonly used dyes include bromophenol blue, xylene cyanol, phenol red, and Orange G. When choosing a loading buffer, pay attention to the apparent migration of the dye(s) (Figure 8BTables 7, 8, and 9) to avoid masking the nucleic acid bands of interest, especially if they have similar molecular sizes (Figure 8C) [2]. Dye masking makes analysis and quantitation of the desired bands problematic and less reliable.

Table 7. Apparent molecular sizes of bromophenol blue and xylene cyanol FF in agarose gels of different percentages

 Bromophenol blueXylene cyanol FF
Gel %TBETAETBETAE
0.5 750 bp1,150 bp13,000 bp16,700 bp
0.6540 bp850 bp8,820 bp11,600 bp
0.7410 bp660 bp6,400 bp8,500 bp
0.8320 bp530 bp4,830 bp6,500 bp
0.9260 bp440 bp3,770 bp5,140 bp
1.0220 bp370 bp3,030 bp4,160 bp
1.2160 bp275 bp2,070 bp2,890 bp
1.5110 bp190 bp1,300 bp1,840 bp
2.065 bp120 bp710 bp1,040 bp
3.030 bp60 bp300 bp460 bp
4.018 bp40 bp170 bp260 bp
5.012 bp27 bp105 bp165 bp

Table 8. Apparent molecular sizes of bromophenol blue and xylene cyanol FF in polyacrylamide gels of different percentages run with denaturing buffers

Acrylamide:Bis (19:1), gel %Bromophenol blueXylene cyanol FF
4.050 bases230 bases
5.035 bases130 bases
6.026 bases105 bases
8.019 bases75 bases
10.012 bases55 bases
15.010 bases40 bases
20.08 bases28 bases
30.06 bases20 bases

 

Table 9. Apparent molecular sizes of bromophenol blue and xylene cyanol FF in polyacrylamide gels of different percentages run with non-denaturing buffers

Acrylamide:Bis (19:1), gel %Bromophenol blueXylene cyanol FF
3.5100 bp460 bp
5.065 bp260 bp
8.045 bp160 bp
12.020 bp70 bp
15.015 bp60 bp

Table 7. Apparent molecular sizes of bromophenol blue and xylene cyanol FF in agarose gels of different percentages

 Bromophenol blueXylene cyanol FF
Gel %TBETAETBETAE
0.5 750 bp1,150 bp13,000 bp16,700 bp
0.6540 bp850 bp8,820 bp11,600 bp
0.7410 bp660 bp6,400 bp8,500 bp
0.8320 bp530 bp4,830 bp6,500 bp
0.9260 bp440 bp3,770 bp5,140 bp
1.0220 bp370 bp3,030 bp4,160 bp
1.2160 bp275 bp2,070 bp2,890 bp
1.5110 bp190 bp1,300 bp1,840 bp
2.065 bp120 bp710 bp1,040 bp
3.030 bp60 bp300 bp460 bp
4.018 bp40 bp170 bp260 bp
5.012 bp27 bp105 bp165 bp

Table 8. Apparent molecular sizes of bromophenol blue and xylene cyanol FF in polyacrylamide gels of different percentages run with denaturing buffers

Acrylamide:Bis (19:1), gel %Bromophenol blueXylene cyanol FF
4.050 bases230 bases
5.035 bases130 bases
6.026 bases105 bases
8.019 bases75 bases
10.012 bases55 bases
15.010 bases40 bases
20.08 bases28 bases
30.06 bases20 bases

 

Table 9. Apparent molecular sizes of bromophenol blue and xylene cyanol FF in polyacrylamide gels of different percentages run with non-denaturing buffers

Acrylamide:Bis (19:1), gel %Bromophenol blueXylene cyanol FF
3.5100 bp460 bp
5.065 bp260 bp
8.045 bp160 bp
12.020 bp70 bp
15.015 bp60 bp

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).

3. Running the electrophoresis gel

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.

Horizontal gels should be oriented in a gel box such that sample wells are on the side of the negative electrode to move samples towards the positive electrode when electrophoresis starts (Figure 10A). This orientation may be memorized as “run towards the red” since the positive electrode is commonly coded red. Vertical gel boxes are designed with wells located at the top (Figure 10B). 

Running buffer choice

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.

ElectrodeNegative (–)Positive (+)
Electron flowInOut
Chemical reaction4 H2O + 4 e– → 2 H2 (gas) + 4 OH–2 H2O → O2 (gas) + 4 H+ 4 e
pH changesBasicAcidic

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

BufferAdvantagesDisadvantagesNucleic acid resolution
DNARNA
TAE
  • Better separation of large fragments
  • Low buffering capacity; better for shorter runs
  • Good for downstream enzymatic applications

More prone to overheating

>1,000 bp>1,500 bases
TBE
  • Better for shorter fragments (linear dsDNA migrates ~10% slower in TBE)
  • High ionic strength and buffering capacity; good for longer runs
  • Less prone to overheating

Inhibits enzymes, making it unsuitable for downstream enzymatic steps (e.g., cloning)

<5,000 bp<1,500 bases

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.

Voltage

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

The voltage may be adjusted according to the size of the DNA fragments to be separated, as well as the type of running buffer used (Table 12) [2]. Recommended voltages are usually provided with commercially available nucleic acid ladders, for optimal separation of the fragments in each product. Low voltage typically slows the migration of the nucleic acids, which may result in diffusion of small molecules and poor resolution (Figure 12A). On the other hand, when the voltage is too high, poor separation and smearing of samples may occur; in some instances, overheating of the buffer produces “smile bands,” and samples may be denatured (Figure 12B).

Table 12. Run conditions based on fragment size

Size of the DNAVoltageOptimal running buffer
<1 kb5–10 V/cmTBE
1–5 kb4–10 V/cmTAE or TBE
>5 kb1–3 V/cmTAE
Up to 10 kbUp to 23 V/cmTAE

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.

Run time

The length of the gel, the voltage used, and the sizes of the molecules in the sample will determine the time needed for the gel run. Usually, electrophoresis is run until the band of interest has migrated 40–60% of the gel length. At specific time intervals during the gel run, the relative positions of the loading dyes are monitored, until the bromophenol blue dye has migrated approximately 60% of the gel length and/or the Orange G dye has migrated 80% of the gel length. Most importantly, run time should be monitored to help ensure the smallest molecules in the samples or standards do not migrate off the gel. Run times shorter than necessary will not be sufficient to completely resolve the bands (Figure 13). DNA ladders containing tracking dyes that run behind and ahead of the samples are available to help monitor gel runs, as well as to help ensure that bands of interest are not masked by the dyes.

4. Visualizing gel electrophoresis results

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.

Table 13. Common nucleic acid gel stains and calorimetric detection

StainBenefits and considerationsSensitivity for detection
(approximate dsDNA amounts)
  • Methylene blue
  • Crystal violet
  • Requires no special equipment
  • Requires a destaining step
  • Low sensitivity

0.5–1 µg

Table 14. Common nucleic acid gel stains and fluorescent detection

StainBenefits and considerationsSensitivity for detection
(approximate dsDNA amounts)
  • Requires excitation by UV and/or blue light
  • Often mutagenic
  • May require special waste disposal
  • Offers shorter workflows with high sensitivity
25 pg–1 ng

Table 15. Common nucleic acid gel stains and radioactive detection

StainBenefits and considerationsSensitivity for detection
(approximate dsDNA amounts)
  • 32P
  • 33P
  • 3H
  • Requires training and precautions for radioactive safety
  • Detects or labels samples by radioactive probes or nucleotides
  • Common for detection of oligonucleotides
  • Considered most sensitive
10 fg–1 ng

Table 13. Common nucleic acid gel stains and calorimetric detection

StainBenefits and considerationsSensitivity for detection
(approximate dsDNA amounts)
  • Methylene blue
  • Crystal violet
  • Requires no special equipment
  • Requires a destaining step
  • Low sensitivity

0.5–1 µg

Table 14. Common nucleic acid gel stains and fluorescent detection

StainBenefits and considerationsSensitivity for detection
(approximate dsDNA amounts)
  • Requires excitation by UV and/or blue light
  • Often mutagenic
  • May require special waste disposal
  • Offers shorter workflows with high sensitivity
25 pg–1 ng

Table 15. Common nucleic acid gel stains and radioactive detection

StainBenefits and considerationsSensitivity for detection
(approximate dsDNA amounts)
  • 32P
  • 33P
  • 3H
  • Requires training and precautions for radioactive safety
  • Detects or labels samples by radioactive probes or nucleotides
  • Common for detection of oligonucleotides
  • Considered most sensitive
10 fg–1 ng

Figure 14. Different types of nucleic acid-binding dyes and where they bind or intercalate in DNA. Dye molecules include intercalators, major groove binders, external binders, minor groove binders, and bis-intercalators.

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.

In-gel and post-electrophoresis staining methods

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

MethodsBenefitsConsiderations
In-gel staining
  • More convenient
  • Faster workflow
  • Requires less stain
  • Stain may run off after a long run
  • Stain can be used only once
  • May alter sample mobility
Post-electrophoresis staining
  • Provides more accurate analyses of molecular sizes
  • Allows reuse of staining solutions or use with multiple gels
  • Longer workflow
  • Requires more stain
  • May amount to more hazardous waste

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.

Figure 15. Differences in DNA migration between in-gel vs. post-electrophoresis staining. (A) Ethidium bromide incorporated in the gel moves in the direction opposite of nucleic acids. The yellow line marks the border between the fluorescing background ethidium bromide and the area of the gel where the dye has been depleted. (B) A large intercalating dye, when included in the gel as opposed to applied post-electrophoresis, affects the migration of sample bands. To illustrate the effect, two ladders were run side by side in two gels. The high molecular weight ladder (lane 1) migrated similarly in both gels (blue lines mark reference bands that show comparable band positions). The low molecular weight ladder (lane 2) migrated differently in the presence of the dye during electrophoresis.

UV shadowing

Instead of staining with dyes, nucleic acids may be indirectly visualized by a method called UV shadowing, which takes advantage of the property of UV absorption by nucleic acids. It is commonly used for separation and purification of oligonucleotides and RNA by electrophoresis, where simple detection is sufficient and/or use of intercalating dyes could impact downstream applications.

For detection by UV shadowing, nanograms to micrograms of samples are needed, and a thin and transparent gel like polyacrylamide should be used to help ensure UV absorption and transmission. In a UV shadowing protocol, the gel is removed from the cassette after electrophoresis to maximize detection, wrapped in clear plastic film for protection, and then placed on a UV-fluorescent thin layer chromatography (TLC) plate. When the gel is exposed to UV radiation, absorption by the nucleic acid bands casts shadows on the TLC plate (Figure 16). The shadowy areas of the gel of desired sizes are cut out for further processing. 

5. Documenting gel electrophoresis results

Fluorescence imaging

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

Epi-illuminatorTransilluminator
  • An excitation light source is placed above the gel
  • Samples are subjected to less energy because light source is farther from sample
  • Less UV damage
  • Lower signal strength from gel bands
  • An excitation light source is placed below the gel
  • Samples are subjected to more energy because due of proximity to sample
  • May increase UV-induced damage
  • Higher signal strength from gel bands

Autoradiography

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. 

Integrated imaging

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.

References
  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.
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