Nucleic Acid Electrophoresis Troubleshooting Guide

1. No or poorly visible bands

Possible causes	Recommendations
Gel preparation
Low quantity of sample	Make sure sufficient amounts of sample are loaded onto the gel. Gel electrophoresis typically requires nanograms of sample, per band, to visualize; thus, 0.1–0.2 μg of sample per millimeter of a gel well’s width is generally recommended. Use a gel comb with deep, narrow wells for improved visualization of low-quantity samples.
Sample degraded	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.
Loading dye masking the desired band	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 they are in low quantity.
Gel run
Gel over-run	Monitor the run time and migration of loading dyes to avoid running smaller sample molecules off the gel.
Electrodes reversed	Ensure the electrodes are connected correctly to the power supply. The gel wells should be on the same side as the negative electrode when setting up a horizontal gel.
Sample visualization
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 time 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 longer staining times for fluorescent stains to penetrate. Alternatively, consider stains with faster penetration properties.
High background of stain	Destain the gel, or choose a stain with low intrinsic fluorescence to circumvent destaining.
Uneven staining	If samples are only partially visible or visible only in some lanes of the gel: For in-gel staining, mix the stain thoroughly throughout the agarose solution (without creating bubbles) when preparing the gel. For post-electrophoresis staining, ensure that the gel is fully submerged in the staining solution, with gentle shaking for a sufficient time period.
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.

2. Smeared or diffuse (fuzzy) bands

Possible causes	Recommendations
Gel preparation
Thick gel	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	Clean the gel comb properly before using it in casting the gel. 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
Sample overloaded	Use no more than necessary amounts of samples in gel electrophoresis; 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 degraded	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	Protein present in the sample may interfere with sample mobilities in the gel. Remove the protein by purifying the sample, or dissociate/denature the protein by preparing the sample in a loading dye with SDS and heating it before loading.
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
Bubbles introduced during sample loading	Make sure air bubbles are not trapped in the well during sample loading, to avoid band distortion.
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 become 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
Band diffusion	Avoid gel storage or a long delay between completion of electrophoresis and visualization of the gel. Bands of smaller molecular sizes, as well as the nucleic acid stain included in the gel, may become 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 diffuse 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.

3. Poorly separated bands

Possible causes	Recommendations
Gel preparation
Incorrect gel percentage	Ensure that the gel percentage is appropriate for resolving the desired sizes of samples. Smaller molecular sizes require higher gel percentages. When preparing agarose gels, adjust the gel volume with water after boiling to compensate for evaporation and to prevent the gel being of a higher 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 (e.g., 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
Sample overloaded	Use no more than necessary amounts of a sample in gel electrophoresis; 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	Protein present in the sample may interfere with sample mobilities in the gel. Remove the protein by purifying the sample, or dissociate/denature the protein by preparing the sample in a loading dye with SDS and heating before loading.
Incompatible loading buffer	For electrophoresis of single-stranded nucleic acids (e.g., RNA), use a loading buffer 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.
Low volume of sample	Make sure sample volume fills at least 30% of the well to avoid band distortion.
Gel run
Bubbles introduced during sample loading	Make sure air bubbles are not trapped in the well during sample loading, to avoid band distortion.
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 become 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 2 hours.

4. Anomalous separation or migration

Possible causes	Recommendations
Gel preparation
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 for pouring the gel and inserting the comb. The comb should be perfectly 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
Sample containing different conformations	Different conformations (e.g., supercoiled, linear, relaxed/nicked) of plasmid DNA display different mobilities in electrophoresis. In electrophoresis of plasmid DNA, use no more of an intercalating dye than necessary, as it may change the plasmid’s conformation. When working with single-stranded nucleic acids like RNA, include a denaturant in the sample buffer and heat the sample prior to gel loading, to maintain single-strandedness.
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 (e.g., methylated, or labeled with biotin or large fluorescent molecules) can migrate more slowly than unmodified DNA of the same base pair length.
Sample with cohesive ends	Use a loading buffer with SDS and heat the sample, to prevent nucleic acids with long complementary ends from interacting with each other and forming concatemers.
Proteins bound to nucleic acids	Use a loading buffer with SDS and heat the sample, to disrupt the interaction between proteins and nucleic acids, for example, after restriction digestion and ligation.
Gel run
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 2 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 generated	Ensure that the running buffer has high buffering capacity, circulate/replenish the buffer during a long run (>2 hr), and cool the gel apparatus when appropriate. Lower the voltage, or set the current or power constant.
Sample visualization
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.

5. Incorrect quantitation data

Possible causes	Recommendations
Sample preparation
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
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.

6. Other gel electrophoresis issues

a. Sample remains in the gel well

Possible causes	Recommendations
Sample preparation
Overloaded sample	Use no more than necessary amount of samples 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 them 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 rise from the submerged electrodes in the electrophoresis chamber when electrophoresis begins (indicating 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.

b. Sequence mutations after electrophoresis

Possible causes	Recommendations
Sample visualization
Radiation damage	As much as possible, minimize exposure of DNA to UV light. Use UV light of a longer wavelength (e.g., 360 nm) instead of a shorter wavelength (e.g., 254–312 nm) to visualize nucleic acids in gels. Alternatively, use stains with less damaging, long excitation wavelengths to visualize the DNA. Consider epi-illumination instead of transillumination during visualization.

c. Sample floats after loading

Possible causes	Recommendations
Sample preparation
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.

d. Speckles in the gel

Possible causes	Recommendations
Sample visualization
Fluorescing contaminants	Dust and/or certain microorganisms in the gel may fluoresce and produce speckles. Use molecular biology–grade reagents and clean, dedicated labware to prepare gels.