In the nucleic acid electrophoresis workflow, a number of steps need to be performed, each of which could impact the outcome of nucleic acid separation, as discussed in the previous section covering experimental setup. Topics on this page address 7 additional considerations associated with samples, reagents, and running parameters, including:

  1. Nucleic acid sequence and conformation
  2. Gel reagent properties
  3. Gel thickness and well sizes
  4. Running buffer types
  5. Voltage, current, and power in gel runs
  6. Nucleic acid stain properties
  7. Gel staining approaches

1. Sequence and conformation of nucleic acid samples

The basic principles of electrophoresis imply that nucleic acid samples have different rates of mobility when they are of different sizes. However, nucleic acids with the same number of nucleotides but different sequence composition and conformation may have different mobilities during electrophoresis (Figure 1).

  • Sequence: AT-rich DNA may migrate more slowly than GC-rich DNA of the same size, especially in high-resolution electrophoresis. Similarly, DNA molecules with 4–6 adenosine repeats at approximately every 10 bp (called curved DNA) will migrate irregularly, especially in polyacrylamide gels [1,2]. Their anomalous migration is likely due to sequence composition affecting their molecular conformation.
  • Conformation: The migration of DNA molecules of the same sequence but differing conformations, such as circular and linearized plasmids, is affected by the compactness of each conformation as they move through the gel pores. Highly compact supercoiled molecules migrate the fastest, followed by flexible linear and open circular molecules (Figure 1). This differential migration may be exploited to examine the integrity of plasmid DNA after isolation, since intact plasmid DNA is desirable in applications like transfection of mammalian cells for gene overexpression.

Figure 1. Electrophoretic migration of the same DNA in various conformations. (A) Electrophoresis of nicked circular, linear, and supercoiled plasmid DNA. (B) Conformation of relaxed circular, linear, and supercoiled plasmid DNA. Nicked plasmids assume a relaxed, open circular conformation and take up the most volume, migrating most slowly through the gel; linearized plasmids move through the gel at a slightly higher rate; intact, supercoiled plasmids, being the most compact, migrate the fastest.

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2. Properties of agarose and acrylamide reagents to consider for electrophoresis

The size and desired resolution of nucleic acid samples to be separated will often drive selection between agarose or polyacrylamide gels (learn more about the two gel choices). In general, higher gel percentages are more effective for resolving smaller molecules. Table 1 summarizes properties to consider when selecting agarose and acrylamide gel reagents for nucleic acid electrophoresis.

Table 1. Properties of agarose and acrylamide gel reagents relevant to electrophoresis [3,4].

Property Implications
Clarity Agarose forms translucent gels. Therefore, agarose with a higher clarity specification ensures minimal fluorescence background during visualization and documentation of the gel.
Electroendosmosis (EEO) During electrophoresis, the movement of buffer towards an electrode may be affected by the interaction between buffer ions and charge molecules within and on the surface of the agarose matrix, in a process known as electroendosmosis or EEO. Since positively charged ions (cations) of the buffer flow in the direction opposite of nucleic acids (Figure 2A), agarose with higher negative charges can impact movement of the cations and therefore the separation of large nucleic acids (>10 kb).
The EEO value can be regarded as an indirect indicator of the amount of negatively charged groups in the agarose. Oxygen atoms on the side chains of agarose (indicated by X in Figure 2B) may carry hydrogens (X = H), or negatively charged groups such as sulfate (SO3) and pyruvate (CH3COCOO). Since negative charges attract buffer cations, the presence of these groups on agarose lowers the efficiency and resolution of nucleic acid separation.
Gel point Gel point indicates the temperature at which an agarose solution forms a gel. The higher the gel percentage, the higher the gel point.
Gel strength Gel strength, expressed in the unit of force (g/cm2), corresponds to the ability of a gel to withstand breakage and is dependent upon agarose concentration. The higher the gel strength, the easier it is to handle.
Genetic quality Genetic quality (GQ) indicates whether agarose is suitable for molecular biology applications, based on levels of contaminants and enzyme inhibitors.
Melting point Melting point is the temperature at which agarose melts. Since gelled agarose melts when heat is applied, the melting point is always higher than the gel point. Low melting point (LMP) agarose is a specific type of agarose that melts at a significantly lower temperature (~25°C) than standard agarose. LMP agarose also exhibits a lower gel point, which is useful for extraction of large nucleic acids and setting up in-gel enzymatic reactions like ligation.
Property Implications
Molecular biology grade Use high-quality, molecular biology–grade reagents that have been tested for nuclease activity and the presence of contaminants. This will protect the integrity of nucleic acid samples during electrophoresis.
Stability/shelf life Commercially prepared stock solutions of polyacrylamide are often stabilized by infusion with a gas to prolong their stability. If you prepare your own polyacrylamide stock solutions in your lab, they should be used within a few months, as they break down to acrylic acid over time. Acrylamide and bisacrylamide, in powder or solution, should be stored in dark containers to protect them from light.

The ammonium sulfate (APS) solution is best prepared fresh, for free radical formation to initiate gel polymerization. The prepared solution may be stored at 4°C for about one month, but its efficiency decreases over time.

Tetramethylethylenediamine (TEMED), a reagent that stabilizes the free radicals formed in gel polymerization, should be stored tightly capped to prevent oxidation.
Total percentage of monomers, w/v (%T) The total percentage of monomeric acrylamide and crosslinking bisacrylamide in solution (%T) determines the pore size of a polyacrylamide gel. For example, a 10% polyacrylamide gel is composed of 10% (w/v) of acrylamide and bisacrylamide. The higher the %T, the smaller the pore size and higher the resolving power to separate smaller molecules (learn more: recommended % of polyacrylamide gels).
Percentage of crosslinker (%C) The %C refers to the amount of crosslinker with respect to the total amount of monomers (w/w). At a given %T, the higher the %C, the smaller the pore sizes. %C may also be presented as the ratio of acrylamide to bisacrylamide (e.g., 5 %C as 19:1). Polyacrylamide gels of 5 %C (19:1) and 3.3 %C (29:1) are most commonly used in nucleic acid electrophoresis.
Property Implications
Clarity Agarose forms translucent gels. Therefore, agarose with a higher clarity specification ensures minimal fluorescence background during visualization and documentation of the gel.
Electroendosmosis (EEO) During electrophoresis, the movement of buffer towards an electrode may be affected by the interaction between buffer ions and charge molecules within and on the surface of the agarose matrix, in a process known as electroendosmosis or EEO. Since positively charged ions (cations) of the buffer flow in the direction opposite of nucleic acids (Figure 2A), agarose with higher negative charges can impact movement of the cations and therefore the separation of large nucleic acids (>10 kb).
The EEO value can be regarded as an indirect indicator of the amount of negatively charged groups in the agarose. Oxygen atoms on the side chains of agarose (indicated by X in Figure 2B) may carry hydrogens (X = H), or negatively charged groups such as sulfate (SO3) and pyruvate (CH3COCOO). Since negative charges attract buffer cations, the presence of these groups on agarose lowers the efficiency and resolution of nucleic acid separation.
Gel point Gel point indicates the temperature at which an agarose solution forms a gel. The higher the gel percentage, the higher the gel point.
Gel strength Gel strength, expressed in the unit of force (g/cm2), corresponds to the ability of a gel to withstand breakage and is dependent upon agarose concentration. The higher the gel strength, the easier it is to handle.
Genetic quality Genetic quality (GQ) indicates whether agarose is suitable for molecular biology applications, based on levels of contaminants and enzyme inhibitors.
Melting point Melting point is the temperature at which agarose melts. Since gelled agarose melts when heat is applied, the melting point is always higher than the gel point. Low melting point (LMP) agarose is a specific type of agarose that melts at a significantly lower temperature (~25°C) than standard agarose. LMP agarose also exhibits a lower gel point, which is useful for extraction of large nucleic acids and setting up in-gel enzymatic reactions like ligation.
Property Implications
Molecular biology grade Use high-quality, molecular biology–grade reagents that have been tested for nuclease activity and the presence of contaminants. This will protect the integrity of nucleic acid samples during electrophoresis.
Stability/shelf life Commercially prepared stock solutions of polyacrylamide are often stabilized by infusion with a gas to prolong their stability. If you prepare your own polyacrylamide stock solutions in your lab, they should be used within a few months, as they break down to acrylic acid over time. Acrylamide and bisacrylamide, in powder or solution, should be stored in dark containers to protect them from light.

The ammonium sulfate (APS) solution is best prepared fresh, for free radical formation to initiate gel polymerization. The prepared solution may be stored at 4°C for about one month, but its efficiency decreases over time.

Tetramethylethylenediamine (TEMED), a reagent that stabilizes the free radicals formed in gel polymerization, should be stored tightly capped to prevent oxidation.
Total percentage of monomers, w/v (%T) The total percentage of monomeric acrylamide and crosslinking bisacrylamide in solution (%T) determines the pore size of a polyacrylamide gel. For example, a 10% polyacrylamide gel is composed of 10% (w/v) of acrylamide and bisacrylamide. The higher the %T, the smaller the pore size and higher the resolving power to separate smaller molecules (learn more: recommended % of polyacrylamide gels).
Percentage of crosslinker (%C) The %C refers to the amount of crosslinker with respect to the total amount of monomers (w/w). At a given %T, the higher the %C, the smaller the pore sizes. %C may also be presented as the ratio of acrylamide to bisacrylamide (e.g., 5 %C as 19:1). Polyacrylamide gels of 5 %C (19:1) and 3.3 %C (29:1) are most commonly used in nucleic acid electrophoresis.

Figure 2. (A) Movement of buffer cations relative to nucleic acids during electrophoresis. (B) Structure of an agarose unit with positions on oxygen that may carry negatively charged groups (indicated by X).

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3. Gel thickness and well sizes

Gel thickness and well size may also influence electrophoresis results, with both agarose and polyacrylamide gels [3].

In general, thicker gels may cause bands to diffuse, due to more heat generated during gel runs. Suboptimal visualization may also occur due to a high background of gel stain or the length of time needed to stain and/or destain the gel (if post-electrophoresis staining is performed). For agarose gels, a thickness of 3–4 mm generally works well, and gels thicker than 5 mm are not recommended. The thickness of polyacrylamide gels is defined by spacers for gel casting plates supplied by the manufacturers, the most common of which are 0.75 mm, 1.0 mm, and 1.5 mm.

The size of the well, defined by the shape of the gel comb, affects not only how much sample can be loaded but also resolution of the bands. While larger wells accommodate increased sample loading, they may produce thick bands, reducing band resolution and creating smears. On the other hand, long and narrow wells accommodate smaller sample amounts but often provide sharper and more well-defined bands for better resolution. More compact samples also offer higher band intensity from less input.

4. Running buffer types

Two common running buffers used in nucleic acid electrophoresis are Tris-acetate EDTA (TAE) and Tris-borate EDTA (TBE) [5] (See also the gel running step in the previous section). Lower molecular weight samples (e.g., DNA <1,000 bp) are separated better with TBE buffer, which has high ionic strength and buffering capacity; larger DNA fragments tend to not separate well in TBE buffer. For denaturing electrophoresis, which is used to resolve molecules that tend to form secondary structures, like RNA, TBE buffer is usually used, since 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 (e.g., 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 (it is a relatively less charged buffer), and lowers field strength, all of which decrease the tendency of large molecules to smear [6].

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5. Electrical running parameters: voltage, current, and power

To separate samples by electrophoresis, an electrical field is applied so that negatively charged nucleic acids migrate toward the positive electrode. Hence, electrical parameters governing electrophoresis can impact sample migration and resolution of its constituent fragments [7,8].

The following equations, derived from Ohm’s Law, may be used to express how voltage (V), current (I), and power (P) can influence electrophoresis results.

Voltage = current x resistance, or V = I x R
Power = current x voltage, or P = I x V
Power can be expressed as P = Ix R, since V = I x R.

The resistance (R) during a gel run is intrinsic to the system. For example, buffer (conductivity and buffering capacity), temperature, gel properties (percentage, height, length, number, cross section), etc., of the system all affect the resistance. In a conductive medium, the resistance decreases when the temperature increases, since higher temperatures allow more current flow. Over the course of a gel run, however, the resistance may vary.

Another important contributing factor in electrophoresis is heat. Heat generated is directly proportional to the power consumed by the system and is dependent upon buffer conductivity, applied voltage, and resistance. The higher the conductivity of a buffer (especially when composed of small ions), the more the current flows. Current flow is also enhanced by high voltage and low resistance. The rise in overall current flow increases power and heat generated by the system.

Table 2 describes how resistance and heat contribute to effects of constant voltage, power, and current on an electrophoresis system. Regardless of the electrical parameter that is set constant with a power supply, the voltage should be capped at slightly lower than the maximum value that the system can handle, to avoid overheating and damage to the equipment and samples. A general recommendation is to set electrical running parameters high enough to separate samples efficiently without generating excessive heat. 

Table 2. Voltage, power, and current in electrophoresis.

  Voltage (V) Power (P) Current (I)
Description
  • V = I x R
  • Corresponds to the electrical potential difference between the two electrodes of a gel system
  • P = I x V or P = I2 x R
  • Measures the rate of energy conversion, which is correlated to heat generated by the system
  • I = V/R
  • Denotes the flow of buffer ions and is directly correlated to the applied voltage
Electrophoresis implications
  • Voltage contributes to the field strength (V/cm).
  • Higher voltage moves charged molecules faster.
  • Constant voltage is recommended, as it offers the most control over the speed of sample migration.
  • Variation in the resistance (R) (e.g., from different numbers and cross-sections of gels) in a given system is compensated for by changes in the current (I) at the constant voltage (V = I x R), keeping the rate of sample migration relatively constant.
  • Constant power prevents overheating of the system but may result in variable sample mobility.
  • Depletion of buffer ions (decreased current) over a lengthy gel run may result in a progressive increase in voltage to maintain constant power (P = I x V).
  • Isoelectric focusing (IEF) of proteins represents an application of constant power, where a gradual increase in voltage is desired to “focus” samples into narrow zones at the completion of gel runs.
  • Current contributes to power (P = I2 x R), by the order of magnitude of two.
  • Constant current keeps power consumption and heat generation of the system relatively constant (in continuous nongradient gels for nucleic acids).
  • In discontinuous or gradient gel electrophoresis for proteins, constant current may be useful to stack samples. Since I = V/R, when samples enter higher-percentage gels (having increased resistance), the voltage also increases to keep the current constant, exerting a larger electrical force on the samples in the process.

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6. Common properties of fluorescent dyes for nucleic acids

Fluorescent dyes are often used to stain nucleic acid samples in electrophoresis for visualization. In addition to sensitivity, characteristics of the dyes such as excitation wavelength, binding affinity, and rate of gel penetration can impact workflow and applications of electrophoresis (Table 3) [9].

Table 3. Common properties of fluorescent dyes used in nucleic acid staining.

Property Implications
Binding affinity The binding affinity of a dye is an important factor because fluorescent enhancement is often observed upon dye binding to the samples. In general, nucleic acid dyes have higher affinity for double-stranded molecules (e.g., DNA) than single-stranded molecules (e.g., RNA), since it is easier to bind to double-stranded helices. For RNA electrophoresis, unique dyes with higher affinity for single-stranded molecules can increase specificity and sensitivity in RNA detection.
Compatibility with denaturants Urea and formamide are typically used as denaturants in RNA electrophoresis. Dyes that are resistant to quenching by these denaturants should be considered in denaturing electrophoresis, for improved effectiveness. Otherwise, denaturing gels should be washed to remove denaturants prior to staining.
Dynamic range The dynamic range represents the orders of magnitude in which linear detection of sample amounts occurs. Therefore, dyes with a broader dynamic range allow more accurate quantitation of bands in the gel.
Excitation wavelength Longer wavelengths exert lower energy, meaning less damage to nucleic acids. Thus, dyes excited using blue light protect sample integrity better than those excited by UV light. Such effects can have pronounced implications in downstream applications, such as cloning efficiency.
Gel penetration Dyes that penetrate gels faster not only shorten the workflow but also stain thick and high-percentage gels better when used post-electrophoresis.
Intrinsic fluorescence Dyes with low intrinsic fluorescence result in lower background in gel staining, circumventing the need to destain while improving detection.
Mutagenicity Fluorescent dyes used in nucleic acid staining are often mutagenic due to their intercalating property. Dyes that have been shown to be less mutagenic and classified as nonhazardous should be considered for added safety and convenient disposal.

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7. Dye binding to samples: 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 two methods each have advantages and challenges, as listed in Table 4.

Table 4. Benefits and considerations of in-gel vs. post-electrophoresis staining.

Methods Benefits Considerations
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 3A). 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 3B). Furthermore, high levels of intercalating dyes included in gels may change the conformation of supercoiled plasmid DNA, altering their mobility (apparent molecular weights) 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, and generates more hazardous waste when using dyes like ethidium bromide.

Figure 3. Results of nucleic acid gel electrophoresis with an intercalating dye. (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.

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In summary, selecting appropriate reagents, parameters, and methods, in addition to the workflow setup, is vital to achieving optimal results in electrophoresis for the separation and analysis of nucleic acids

References

  1. Carrera P, Azorín F (1994) Structural characterization of intrinsically curved AT-rich DNA sequences. Nucleic Acids Res 22(18):3671–3680.
  2. Stellwagen NC (2009) Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution. Electrophoresis 30 Suppl 1:S188–195.
  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. Chory J, Pllard JD (1999) Resolution and Recovery of Small DNA Fragments. In: Ausubel FM et al. (editors) Current Protocols in Molecular Biology. Supplementary 45: Unit 2.7.1–2.7.8.
  5. Brody JR, Kern SE (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem 333(1):1–13.
  6. 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):1542–1547.
  7. Thermo Fisher Scientific Inc. (2015) Protein Gel Electrophoresis Technical Handbook.
  8. Sheehan D (2009) Electrophoresis. In: Physical Biochemistry: Principles and Applications. West Sussex: Wiley. pp 147–198.
  9. 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.
  10. Sigmon J, Larcom LL (1996) The effect of ethidium bromide on mobility of DNA fragments in agarose gel electrophoresis. Electrophoresis 17(10):1524–1527.
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