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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.
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.
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:
Agarose | Polyacrylamide | |
---|---|---|
Source | Polysaccharide polymer from red algae | Synthetic |
Gel formation | Dissolves in water and forms extensive hydrogen bonds | Polymerizes in the presence of crosslinking agent bis-acrylamide |
Gel casting | Melt and solidify | Initiate chemical reactions |
Nucleic acid recovery | Melt and extract | Dissolve, and diffuse or electroelute |
DNA separation range | 50–50,000 bp | 5–3,000 bp |
Resolving power | 5–10 nucleotides | Single nucleotide |
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:
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.
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 percentage | Range of efficient separation (bp) |
---|---|
0.5 | 2,000–50,000 |
0.6 | 1,000–20,000 |
0.7 | 800–12,000 |
0.8 | 800–10,000 |
0.9 | 600–10,000 |
1.0 | 400–8,000 |
1.2 | 300–7,000 |
1.5 | 200–3,000 |
Table 3. Range of efficient separation in high percentage gels (2–5%)
Gel percentage | Range of efficient separation (bp) |
---|---|
2.0 | 100–2,000 |
3.0 | 25–1,000 |
4.0 | 10–500 |
5.0 | 10–300 |
Table 2. Range of efficient separation in low percentage gels (0.5–1.5%)
Gel percentage | Range of efficient separation (bp) |
---|---|
0.5 | 2,000–50,000 |
0.6 | 1,000–20,000 |
0.7 | 800–12,000 |
0.8 | 800–10,000 |
0.9 | 600–10,000 |
1.0 | 400–8,000 |
1.2 | 300–7,000 |
1.5 | 200–3,000 |
Table 3. Range of efficient separation in high percentage gels (2–5%)
Gel percentage | Range of efficient separation (bp) |
---|---|
2.0 | 100–2,000 |
3.0 | 25–1,000 |
4.0 | 10–500 |
5.0 | 10–300 |
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.
Figure 4. Polyacrylamide formation with acrylamide and bisacrylamide structures. Bisacrylamide is two units of acrylamide connected by a methylene bridge (red).
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 |
---|---|
4.0 | 100–500 bases |
5.0 | 70–400 bases |
6.0 | 40–300 bases |
8.0 | 30–200 bases |
10.0 | 20–100 bases |
15.0 | 10–50 bases |
20.0 | 5–30 bases |
30.0 | 1–10 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) |
---|---|
3.5 | 100–1,000 |
5.0 | 80–500 |
8.0 | 60–400 |
12.0 | 50–200 |
15.0 | 25–150 |
20.0 | 5–100 |
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 |
---|---|
4.0 | 100–500 bases |
5.0 | 70–400 bases |
6.0 | 40–300 bases |
8.0 | 30–200 bases |
10.0 | 20–100 bases |
15.0 | 10–50 bases |
20.0 | 5–30 bases |
30.0 | 1–10 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) |
---|---|
3.5 | 100–1,000 |
5.0 | 80–500 |
8.0 | 60–400 |
12.0 | 50–200 |
15.0 | 25–150 |
20.0 | 5–100 |
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 | %C | Relative pore sizes | Applications |
---|---|---|---|
19:1 | 5% | Small | DNA and denaturing gels |
29:1 | 3.3% | Medium | ssDNA and RNA in nondenaturing gels |
37.5:1 | 2.7% | Large | Protein 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.
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:
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:
Learn more on the effects of structure and conformation on sample mobility
Figure 5. Some loading dyes contain tracking dyes that migrate at different lengths and produce shadows when visualizing the gel. Ladder #2 consists of chromatographically purified DNA fragments in an optimal loading buffer, resulting in bands of equal or desired intensity, lack of band smears, and absence of dye shadows. In contrast, ladder #1 was manufactured with an older technology and loaded in a buffer with suboptimal composition.
Figure 6. Differences in fragment composition of DNA ladders of the same description from vendors A and B. The two left lanes contain 1 kb ladders and the two right lanes contain 100 bp ladders. Because different vendors use a different composition of DNA fragments, the ladder description from vendor to vendor may be different.
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).
Figure 7. Suboptimal sample separation.(A) The amount loaded affects band resolution. (B) A low volume of loaded sample may result in distorted bands.
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).
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:
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 8B, Tables 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.
Bromophenol blue | Xylene cyanol FF | |||
---|---|---|---|---|
Gel % | TBE | TAE | TBE | TAE |
0.5 | 750 bp | 1,150 bp | 13,000 bp | 16,700 bp |
0.6 | 540 bp | 850 bp | 8,820 bp | 11,600 bp |
0.7 | 410 bp | 660 bp | 6,400 bp | 8,500 bp |
0.8 | 320 bp | 530 bp | 4,830 bp | 6,500 bp |
0.9 | 260 bp | 440 bp | 3,770 bp | 5,140 bp |
1.0 | 220 bp | 370 bp | 3,030 bp | 4,160 bp |
1.2 | 160 bp | 275 bp | 2,070 bp | 2,890 bp |
1.5 | 110 bp | 190 bp | 1,300 bp | 1,840 bp |
2.0 | 65 bp | 120 bp | 710 bp | 1,040 bp |
3.0 | 30 bp | 60 bp | 300 bp | 460 bp |
4.0 | 18 bp | 40 bp | 170 bp | 260 bp |
5.0 | 12 bp | 27 bp | 105 bp | 165 bp |
Acrylamide:Bis (19:1), gel % | Bromophenol blue | Xylene cyanol FF |
---|---|---|
4.0 | 50 bases | 230 bases |
5.0 | 35 bases | 130 bases |
6.0 | 26 bases | 105 bases |
8.0 | 19 bases | 75 bases |
10.0 | 12 bases | 55 bases |
15.0 | 10 bases | 40 bases |
20.0 | 8 bases | 28 bases |
30.0 | 6 bases | 20 bases |
Acrylamide:Bis (19:1), gel % | Bromophenol blue | Xylene cyanol FF |
---|---|---|
3.5 | 100 bp | 460 bp |
5.0 | 65 bp | 260 bp |
8.0 | 45 bp | 160 bp |
12.0 | 20 bp | 70 bp |
15.0 | 15 bp | 60 bp |
Bromophenol blue | Xylene cyanol FF | |||
---|---|---|---|---|
Gel % | TBE | TAE | TBE | TAE |
0.5 | 750 bp | 1,150 bp | 13,000 bp | 16,700 bp |
0.6 | 540 bp | 850 bp | 8,820 bp | 11,600 bp |
0.7 | 410 bp | 660 bp | 6,400 bp | 8,500 bp |
0.8 | 320 bp | 530 bp | 4,830 bp | 6,500 bp |
0.9 | 260 bp | 440 bp | 3,770 bp | 5,140 bp |
1.0 | 220 bp | 370 bp | 3,030 bp | 4,160 bp |
1.2 | 160 bp | 275 bp | 2,070 bp | 2,890 bp |
1.5 | 110 bp | 190 bp | 1,300 bp | 1,840 bp |
2.0 | 65 bp | 120 bp | 710 bp | 1,040 bp |
3.0 | 30 bp | 60 bp | 300 bp | 460 bp |
4.0 | 18 bp | 40 bp | 170 bp | 260 bp |
5.0 | 12 bp | 27 bp | 105 bp | 165 bp |
Acrylamide:Bis (19:1), gel % | Bromophenol blue | Xylene cyanol FF |
---|---|---|
4.0 | 50 bases | 230 bases |
5.0 | 35 bases | 130 bases |
6.0 | 26 bases | 105 bases |
8.0 | 19 bases | 75 bases |
10.0 | 12 bases | 55 bases |
15.0 | 10 bases | 40 bases |
20.0 | 8 bases | 28 bases |
30.0 | 6 bases | 20 bases |
Acrylamide:Bis (19:1), gel % | Bromophenol blue | Xylene cyanol FF |
---|---|---|
3.5 | 100 bp | 460 bp |
5.0 | 65 bp | 260 bp |
8.0 | 45 bp | 160 bp |
12.0 | 20 bp | 70 bp |
15.0 | 15 bp | 60 bp |
Figure 9. Effects of heat and SDS on electrophoresis of samples.(A) RNA ladders in a denaturing buffer were loaded onto the gel without heat treatment. (B) DNA samples from a restriction digest and a ligation reaction were prepared in loading buffer with and without SDS. Samples in SDS were heated before gel loading.
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.
Figure 10. Gel setups in horizontal and vertical electrophoresis systems. Arrows indicate the direction of nucleic acid migration in electrophoresis.
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).
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.
Electrode | Negative (–) | Positive (+) |
---|---|---|
Electron flow | In | Out |
Chemical reaction | 4 H2O + 4 e– → 2 H2 (gas) + 4 OH– | 2 H2O → O2 (gas) + 4 H+ + 4 e– |
pH changes | Basic | Acidic |
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]
Buffer | Advantages | Disadvantages | Nucleic acid resolution | |
---|---|---|---|---|
DNA | RNA | |||
TAE |
| More prone to overheating | >1,000 bp | >1,500 bases |
TBE |
| Inhibits enzymes, making it unsuitable for downstream enzymatic steps (e.g., cloning) | <5,000 bp | <1,500 bases |
Figure 11. Effects of running buffer on electrophoresis. (A) The top portion of the gel was not submerged during electrophoresis. (B) The gel was run in a buffer with lower salt concentrations than recommended, which was different from the buffer used in gel preparation.
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
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).
Size of the DNA | Voltage | Optimal running buffer |
---|---|---|
<1 kb | 5–10 V/cm | TBE |
1–5 kb | 4–10 V/cm | TAE or TBE |
>5 kb | 1–3 V/cm | TAE |
Up to 10 kb | Up to 23 V/cm | TAE |
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.
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.
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.
Stain | Benefits and considerations | Sensitivity for detection (approximate dsDNA amounts) |
---|---|---|
|
| 0.5–1 µg |
Stain | Benefits and considerations | Sensitivity for detection (approximate dsDNA amounts) |
---|---|---|
| 25 pg–1 ng |
Stain | Benefits and considerations | Sensitivity for detection (approximate dsDNA amounts) |
---|---|---|
|
| 10 fg–1 ng |
Stain | Benefits and considerations | Sensitivity for detection (approximate dsDNA amounts) |
---|---|---|
|
| 0.5–1 µg |
Stain | Benefits and considerations | Sensitivity for detection (approximate dsDNA amounts) |
---|---|---|
| 25 pg–1 ng |
Stain | Benefits and considerations | Sensitivity for detection (approximate dsDNA amounts) |
---|---|---|
|
| 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.
Two common approaches to staining nucleic acid samples are:
The advantages and challenges of the two methods are listed in Table 16.
Methods | Benefits | Considerations |
---|---|---|
In-gel staining |
|
|
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.
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.
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.
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).
Epi-illuminator | Transilluminator |
---|---|
|
|
Figure 17. Documenting gel electrophoresis results with epi-illumination and transillumination. (A) Epi-illumination contains two light sources above the gel with a detector between. (B) Transillumination contains one light source below the gel with a detector above.
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.
For Research Use Only. Not for use in diagnostic procedures.