Decorative DNA puzzle

Electrophoresis underpins many molecular biology applications. Therefore, problems in nucleic acid gel electrophoresis hinders downstream applications and hampers experimental workflow; often errors in gel electrophoresis negatively impact the results of an experiment. Common problems encountered in nucleic acid gel electrophoresis and appropriate solutions are discussed in this gel electrophoresis troubleshooting guide.

Common problems during nucleic acid gel electrophoresis

Faint bands in gel electrophoresis

Figure 1. Faint bands in gel electrophoresis.

As the name indicates “faint bands” appear fuzzy and are unclear to visualize (Figure 1).

Gel electrophoresis can sometimes result in faint bands or absence of bands due to incorrect sample preparation, low protein concentration, insufficient electrophoresis conditions, or problems with gel or buffer. Click the accordions for best practices to help minimize faint bands in gel electrophoresis.

Gel preparation to minimize faint bands

Gel preparation recommendations to help minimize faint bands in gel electrophoresis

Possible causes for faint bands in gel electrophoresisRecommendations to avoid or minimize faint bands in gel electrophoresis
Low quantity of sample
  • If you have a low quantity of sample, it can be difficult to visualize the bands. To help ensure clear and visible results, it is recommended to load a minimum of 0.1–0.2 μg of DNA or RNA sample per millimeter of gel well width.
  • Use a gel comb with deep and narrow wells.
Sample degradation
  • Make sure reagents selected are molecular biology grade and labware is free of nucleases. Follow good lab practices such as wearing gloves, preventing nuclease contamination, and working in areas designated for handling nucleic acids, especially when working with RNA.
Loading dye masking the desired band
  • Dyes may mask bands of interest if the dyes have a similar molecular size as that of the nucleic acid. Check the apparent migration size of the loading dye(s) used in electrophoresis.
  • The dye may mask and hide the bands of interest, especially when the sample volume is low.
Gel run to minimize faint bands

Gel run recommendations to help minimize faint bands in gel electrophoresis

Possible causes for faint bands in gel electrophoresisRecommendations to avoid or minimize faint bands in gel electrophoresis
Gel over-run
Reversed electrodes
Band visualization to minimize faint bands

Band visualization recommendations to help minimize faint bands in gel electrophoresis

Possible causes for faint bands in gel electrophoresisRecommendations to avoid or minimize faint bands in gel electrophoresis
Low sensitivity of stain
  • Check the sensitivity of the fluorescent stain used to detect the nucleic acids, as provided by the manufacturer.
  • Use more stain and/or a longer staining duration to visualize single-stranded nucleic acids. Alternatively, consider unique stains with higher affinity for single-stranded molecules to increase specificity and sensitivity in detection.
  • For thick or high-percentage gels, allow a longer staining period for fluorescent stains to penetrate. Alternatively, consider stains with faster penetration properties.
High background of stain
Uneven staining
  • If samples are partially visible or visible only in some lanes of the gel:
    • For in-gel staining: when preparing the gel, mix the stain thoroughly in the agarose solution without creating bubbles.
    • For post-electrophoresis staining: ensure that the gel is fully submerged in the staining solution, with gentle shaking for a sufficient duration.
Incorrect light source
  • If a fluorescent dye is used for staining, check its excitation wavelength to make sure the light source used is optimal for exciting the dye for visualization.

Smearing in gel electrophoresis

Smeared bands, also known as diffused and fuzzy bands, have a blurry appearance (Figure 2). The bands are poorly resolved and overlap with adjacent bands, making it difficult to accurately interpret the results. Click the accordions for best practices to minimize formation of smeared bands in gel electrophoresis.

Figure 2. Smeared bands in gel electrophoresis.

Gel preparation to minimize smearing

Gel preparation to help minimize smearing in gel electrophoresis

Possible causes for smearing in gel electrophoresisRecommendations to minimize smearing in gel electrophoresis
Thick gels
  • Keep the gel thickness around 3–4 mm when casting horizontal agarose gels. Gels thicker than 5 mm may result in band diffusion during electrophoresis.
Poorly formed wells
  • Use clean gel combs when casting gels.
  • To prevent sample leakage through the bottom of the gel and smearing of the sample bands, do not push the comb all the way to the bottom of the horizontal gel.
  • Avoid overfilling the gel tray as this can result in connected wells.
  • Allow sufficient time for the wells to form before removing the comb.
  • Once the gel is solidified, remove the comb carefully and steadily to prevent damage to the wells.
Incorrect gel type
  • For electrophoresis of single-stranded nucleic acids (e.g., RNA), prepare a denaturing gel for efficient separation. On the other hand, avoid using denaturing gels with double-stranded DNA samples.
Sample preparation to minimize smearing

Sample preparation recommendations to help minimize smearing in gel electrophoresis

Possible causes for smearing in gel electrophoresisRecommendations to minimize smearing in gel electrophoresis
Sample overloading
  • Do not overload samples in wells; the general recommendation is 0.1–0.2 μg of sample per millimeter of a gel well’s width. Trailing smears, warped or U-shaped bands, and bands that appear fused are a common characteristic of overloaded gels.
Sample degradation
  • Make sure reagents selected are molecular biology grade and that labware is free of nucleases. Follow good lab practices (e.g., wearing gloves, preventing nuclease contamination, working in designated areas, etc.) in handling nucleic acids, especially when working with RNA.
Sample in high-salt buffer
  • Check that the loading buffer’s salt concentration is compatible with the selected gel. Dilute the loading buffer, if necessary.
  • If the nucleic acid sample is already in a high-salt buffer, dilute the sample in nuclease-free water before adding the loading buffer. If needed, purify or precipitate the nucleic acid sample and resuspend it in nuclease-free water to remove excess salt.
Sample containing high amounts of protein
Incompatible loading buffer
  • For electrophoresis of single-stranded nucleic acids, use a loading dye containing a denaturant and then heat the sample, to prevent formation of undesirable duplexes. 
  • For electrophoresis of double-stranded DNA, avoid a loading dye with denaturant and do not heat the sample, to preserve the duplex structure.
Gel run to minimize smearing

Gel run recommendations to help minimize smearing in gel electrophoresis

Possible causes for smearing in gel electrophoresisRecommendations to minimize smearing in gel electrophoresis
Bubbles introduced during sample loading
  • To avoid band distortion, make sure air bubbles are not trapped in the well during sample loading.
Well damaged during sample loading
  • Avoid puncturing the wells with the pipette tips during sample loading.
Sample wells containing residual acrylamide and/or urea
  • When using polyacrylamide gels, flush residual acrylamide (and urea, in the case of denaturing gels) out of the wells, prior to sample loading.
Very low or high voltage
  • Apply voltage as recommended for the size range of the nucleic acids and the running buffer used. Very low or high voltage can create suboptimal resolution in separation of nucleic acids.
Very short or long run time
  • Run the gel long enough to ensure bands are resolved sufficiently. However, a very long run may generate excessive heat, denature samples, and cause bands to diffuse.
Incompatible running buffer
  • Ensure that the gel preparation and running buffers are compatible and prepared correctly.
  • Use a buffer with high buffering capacity for electrophoresis longer than 2 hours.
Sample visualization to minimize smearing

Sample visualization recommendations to help minimize smearing in gel electrophoresis

Possible causes for smearing in gel electrophoresisRecommendations to minimize smearing in gel electrophoresis
Band diffusion
  • Avoid gel storage or a long delay between completion of electrophoresis and visualization of the gel. Bands of smaller molecular sizes and the nucleic acid stain may diffuse.
Co-migrating bands
  • Use the appropriate gel percentage, voltage, and run time to separate bands of similar molecular sizes. Co-migrating bands often appear as a diffused band that is thick and bright.
Out-of-focus camera
  • Be sure that the camera is in focus if the gel is viewed through the lens to display on a screen.

Poorly separated bands

Figure 3. Poorly separated bands.

Poorly separated bands are characterized by closely stacked bands (Figure 3). The individual bands cannot be differentiated easily and are arranged densely. Click the accordions for best practices to promote proper band separation.

Gel preparation to minimize poor band separation

Gel preparation recommendations to help minimize poor band separation in gel electrophoresis

Possible causes for poor band separationRecommendations to minimize poor band separation
Incorrect gel percentage
  • Ensure that the gel percentage is appropriate for resolving the desired sizes of fragments in the sample. Smaller molecular fragments require higher gel percentages. 
  • When preparing agarose gels, adjust the gel volume with water after boiling to compensate for evaporation; this prevents the increase in gel percentage than intended.
Suboptimal gel choice
  • Choose a gel type that works better to separate the samples. Polyacrylamide gels are recommended for resolving nucleic acids <1,000 bp.
Poorly formed wells
  • Clean the gel comb properly before using it in casting the gel. 
  • To prevent sample leakage through the bottom of the gel, do not push the comb all the way to the bottom of the gel.
  • Avoid overfilling the gel tray, as this can result in connected wells.
  • Allow sufficient time for the wells to form before removing the comb.
  • Once the gel is solidified, remove the comb carefully and steadily to prevent damage to the wells.
Incorrect gel type
  • For electrophoresis of single-stranded nucleic acids such as RNA, prepare a denaturing gel for efficient separation.
  • For electrophoresis of double-stranded DNA, avoid using denaturing gels to preserve the duplex structure.
Sample preparation to minimize poor band separation

Sample preparation recommendations to help minimize poor band separation in gel electrophoresis

Possible causes for poor band separationRecommendations to minimize poor band separation
Sample overloading
  • Do not use more than necessary amounts of a sample; 0.1–0.2 μg of sample per millimeter of a gel well’s width is generally recommended. Trailing smears, warped or U-shaped bands, and bands that appear fused are a common characteristic of overloaded gels.
Sample containing high amounts of protein
Incompatible loading buffer
  • For electrophoresis of single-stranded nucleic acids such as RNA, use a loading buffer containing a denaturant and then heat the sample, to prevent formation of duplex structure.
  • For electrophoresis of double-stranded DNA, to preserve the duplex structure, avoid a loading dye with denaturant and do not heat the sample.
Low volume of sample
  • To avoid band distortion, make sure sample volume fills at least 30% of the well.
Gel run to minimize poor band separation

Gel run recommendations to help minimize poor band separation in gel electrophoresis

Possible causes for poor band separationRecommendations to minimize poor band separation
Bubbles introduced during sample loading
  • To avoid band distortion, make sure air bubbles are not trapped in the well during sample loading.
Well damaged during sample loading
  • Avoid puncturing the wells with the pipette tips during sample loading.
Sample wells containing residual acrylamide and/or urea
  • When using polyacrylamide gels, flush residual acrylamide (and urea, in the case of denaturing gels) out of the wells prior to sample loading.
Very low or high voltage
  • Apply voltage as recommended for the size range of the nucleic acid fragments and the running buffer used. Very low or high voltage can create suboptimal resolution in separation of nucleic acids.
Very short or long run time
  • Run the gel long enough to ensure bands are resolved sufficiently. However, a very long run may generate excessive heat, denature samples, and cause bands to diffuse.
Incompatible running buffer
  • Ensure that the gel preparation and running buffers are compatible and prepared correctly.
  • Make sure the buffer used works well with the samples to be separated. For example:
    • TAE buffer works better for separating large fragments (>1,500 bp) in short runs.
    • TBE is better for shorter fragments (<5,000 bp) but may slow migration of linear dsDNA.
  • Use a buffer with high buffering capacity for electrophoresis longer than two hours.

Anomalous separation or migration

Anomalous separation of bands manifests as irregular band migration pattern, such smiling bands (Figure 4). The main causes of bands “smiling” on a gel are uneven heat distribution and uneven distribution of electric field across gel width.

Figure 4. Smiling bands.

Gel preparation to minimize anomalous migration

Gel preparation recommendations to help minimize anomalous migration in gel electrophoresis

Possible causes for anomalous band separationRecommendations to minimize anomalous migration
Nonhomogeneous gel concentration
  • Make sure to mix the matrix solution thoroughly when preparing the gel. There should not be any undissolved powder or unmelted solids after boiling the agarose solution.
Uneven gels or slanted wells
  • Set the gel tray on an even surface when pouring the gel and inserting the comb. The comb should be vertical and perpendicular to the gel surface, parallel to the gel’s top edge, and stable during gel solidification.
Incorrect gel buffer
  • Make sure the gel is prepared in the same buffer as the running buffer.
Sample preparation to minimize anomalous migration

Sample preparation recommendations to help minimize anomalous migration in gel electrophoresis

Possible causes of anomalous migrationRecommendations to minimize anomalous migration
Sample containing different conformations
  • Different conformations of plasmid DNA such as supercoiled, linear, relaxed, or nicked display different mobilities in electrophoresis.
  • In electrophoresis of plasmid DNA, do not use more than required amounts intercalating dye, as it may change the plasmid’s conformation.
  • To maintain single-strandedness nucleic acids like RNA, include a denaturant in the sample buffer and heat the sample prior to gel loading.
Sample of unusual sequences
  • AT-rich DNA may migrate more slowly in high-resolution electrophoresis.
  • “Curved” DNA, which contains 4–6 adenosine repeats at approximately every 10 bp, migrates irregularly in polyacrylamide gels.
  • Modified DNA such as methylated, or labeled with biotin or large fluorescent molecules tend to migrate more slowly than unmodified DNA of the same base pair length.
Sample with cohesive ends
  • To prevent nucleic acids with long complementary ends from interacting with each other and forming concatemers, use a loading buffer with SDS and heat the sample.
Proteins bound to nucleic acids
Gel run to minimize anomalous migration

Gel run recommendations to help minimize anomalous migration in gel electrophoresis

Possible causes of anomalous migrationRecommendations to minimize anomalous migration
Incompatible running buffer
  • Ensure that the gel and running buffers are compatible and prepared correctly.
  • Make sure the buffer used works well with the samples to be separated. For example:
    • TAE buffer works better for separating large fragments (>1,500 bp) in short runs.
    • TBE is better for shorter fragments (<5,000 bp) but may slow migration of linear dsDNA.
  • Use a buffer with high buffering capacity for electrophoresis longer than two hours.
Very high voltage
  • Avoid using more than the recommended voltage, as it could lead to excessive heat generation, sample denaturation, and “smiling” bands.
Excessive heat generation
  • Ensure that the running buffer has high buffering capacity, circulate or replenish the buffer during a long run (more than two hours), and cool the gel apparatus when appropriate.
  • Lower the voltage, or set the current or power at a constant amperage.
Sample visualization to help minimize anomalous migration

Sample visualization recommendations to help minimize anomalous migration in gel electrophoresis

Possible causes of anomalous migrationRecommendations to minimize anomalous migration
Stain binding to sample
  • Consider post-electrophoresis staining when using a large fluorescent stain, as the binding of the stain to the nucleic acids may shift sample mobilities during electrophoresis.

Incorrect quantitation data

Quantitation is estimating the concentration of nucleic acids in a sample by means of gel electrophoresis. Incorrect quantitation data results from using an incorrect ladder or using different loading dyes for the sample and ladder.

Sample preparation to minimize incorrect quantitation

Sample preparation recommendations to help minimize incorrect quantitation in gel electrophoresis

Possible causes for incorrect quantitation dataRecommendations to help minimize incorrect quantitation data
Incorrect ladder
  • Make sure to use a ladder specifically designed for gel quantitation. This type of ladder contains fragments of known quantity for each band.
Different loading dyes used for sample and ladder
  • Use the same loading dye for both the sample and the ladder to obtain reliable quantitation data.
Sample visualization to help minimize incorrect quantitation

Sample visualization recommendations to help minimize incorrect quantitation in gel electrophoresis

Possible causes for incorrect quantitation dataRecommendations to minimize incorrect quantitation data
Incorrect band of the ladder chosen
  • Compare the sample band of interest with a similarly sized reference band of a quantitative ladder for improved accuracy.
Improper intensity measurement
  • For more precise intensity measurements, subtract the gel background from the measured value of the band of interest. Use the built-in quantitation software of a gel imager, if available.
Uneven staining
  • Ensure that the fluorescent stain is mixed well throughout the gel or the staining solution.
  • Make sure that the gel is immersed completely in the staining solution.
  • Allow longer staining times with thick or high-percentage gels for fluorescent stains to penetrate. Alternatively, consider stains with faster penetration properties.
  • For denaturing gels, wash the gel to remove denaturants that quench fluorescent stains. Alternatively, consider a stain that is resistant to quenching by common denaturants.

Other electrophoresis problems

In addition to the above-mentioned electrophoresis problems, other challenges such as samples remaining in wells, sample floatation, and speckling in gels may occur. Click on the accordions for solutions to these problems.

Sample remains in the gel well

Recommendations to help improve sample migration in gel electrophoresis

Possible causes for sample to remain in gel wellsRecommendations to improve sample migration
Sample preparation
Overloaded sample
  • Do not use more than the necessary amount of sample in gel electrophoresis; 0.1–0.2 μg of sample per millimeter of a gel well’s width is generally sufficient. Overloaded samples may remain in the well.
Protein and cell debris in the sample
  • Protein and cell debris binding to nucleic acids may retard sample migration. Remove the contaminants by purifying the sample, or dissociate/denature contaminants by preparing the sample in a loading dye with SDS and heating it before loading.
Gel run
No power
  • Check that the power supply is on, functional, and connected to the electrophoresis chamber.
  • Check that bubbles arise from the submerged electrodes in the electrophoresis chamber when electrophoresis begins, which indicates gas production at the electrodes as the current flows.
Incorrect running buffer
  • Ensure that the running buffer and gel preparation buffer are compatible, prepared correctly, and conductive.
Sequence mutations after electrophoresis

Recommendations to help prevent sequence mutations after gel electrophoresis

Possible causes of sequence mutations after electrophoresisRecommendations to prevent sequence mutations after electrophoresis
Radiation damage
Sample floats after loading

Recommendations to help prevent sample floatation during gel electrophoresis

Possible causes of sample floatationRecommendations to prevent sample floatation
Incorrect loading buffer
  • Ensure that the loading buffer contains a density ingredient so that samples sink into the wells.
Sample in incompatible solution
  • Purify the sample or resuspend nucleic acids in nuclease-free water after precipitation. Carryover ethanol or extraction solvents may prevent samples from sinking into the wells.
Speckles in the gel

Recommendations to help minimize speckles during gel electrophoresis

Possible causes for speckles in gelRecommendations to minimize speckles in the gel
Fluorescing contaminants
  • Dust and certain microorganisms in the gel may fluoresce and produce speckles. Use molecular biology grade reagents and clean, dedicated labware to prepare gels.
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