Guidelines for troubleshooting nucleic acid electrophoresis

Electrophoresis is an important step in many molecular biology applications. Therefore, problems encountered during nucleic acid electrophoresis may affect your handling of downstream applications and ultimately reduce the workflow efficiency of the experiment. In this section, common problems with nucleic acid gel electrophoresis (described below) are discussed and recommended solutions are presented.

1 No or poorly visible bands

possible reasons	suggestion
gel preparation
Few samples	Make sure you load enough sample into the gel. In gel electrophoresis, a few nanograms of sample are usually required for each band to be visible; Therefore, 0.1-0.2 μg of sample per 1 mm gel well width is generally recommended. Use a gel comb with deep holes to improve visualization of low-volume samples.
Sample degradation	Make sure the reagents chosen are of molecular biology grade and that the lab utensils do not contain nucleases. Follow good laboratory practice when handling nucleic acids (e.g., wear gloves, prevent nuclease contamination, operate in designated areas, etc.), especially when handling RNA.
The loading dye masks the target band	Check the apparent migration size of the loading dyes used in electrophoresis. Because the dye may mask and hide the target band, especially if the sample size is low.
Gel run
Gel over-run	The run time and migration of the loaded dye were monitored to avoid removing smaller sample molecules from the gel.
Electrodes reversed	Make sure the electrodes are properly connected to the power supply. When setting the horizontal gel, the gel hole should be on the same side as the negative pole.
Visualization of samples
Low staining sensitivity	Check the sensitivity of the fluorescent stain provided by the manufacturer for detection of nucleic acids. Increase the amount of dye and/or prolong the dyeing time to make single-stranded nucleic acid color. Alternatively, consider unique staining with higher affinity for single-stranded molecules to improve the specificity and sensitivity of the assay. For thicker or high percentage gels, the staining time should be extended to ensure full penetration of the fluorescent stain. Alternatively, consider using a stain with faster penetration properties.
Hgh staining background	Remove the gel or select stains with low intrinsic fluorescence to avoid decolorization.
Uneven staining	If the sample is only partially visible or only visible in certain lanes on the gel: If the in-gel staining method is used, the stain should be thoroughly mixed with the agarose solution when preparing the gel (to avoid foaming). If post-electrophoresis staining is used, ensure that the gel is completely immersed in the staining solution and gently shake for a certain period of time.
Light source error	If a fluorescent dye is used for staining, check the excitation wavelength to ensure that the light source used is optimal for stimulating the dye for visualization.

2 Smeared or diffuse (fuzzy) bands

possible reasons	suggestion
gel production
Gel is sticky	When filling a horizontal agarose gel, make sure the gel thickness is between 3-4 mm. A gel thickness of more than 5 mm may cause the band to spread during electrophoresis.
poor loading holes formulation	The gel comb should be properly cleaned before gel infusion. To prevent sample leakage from the bottom of the gel and sample strip trailing, it is important not to push the gel comb all the way to the bottom of the horizontal gel. Do not overfill the gel tank, otherwise it will lead to sample hole connection. Allow sufficient time for well formation before removing the gel comb. After the gel has set, remove the gel comb carefully and smoothly to prevent damage to the loading Wells.
Wrong gel type	For electrophoresis of single-stranded nucleic acids (e.g., RNA), denaturing gels for efficient separation should be prepared but for electrophoresis of double-stranded DNA samples denaturing gels should be avoided.
sample production
Sample overload	In gel electrophoresis, do not use excessive samples; A sample dosage of 0.1-0.2 μg per 1 mm gel well width is generally recommended. Trailing, curved or U-shaped bands and fused bands are common manifestations of overload gel.
Sample degradation	Make sure the reagents chosen are of molecular biology grade and that the utensils do not contain nucleases. Follow good laboratory practice when handling nucleic acids (e.g., wear gloves, prevent nuclease contamination, operate in designated areas, etc.), especially when handling RNA.
The samples were dissolved in high salt buffer	Check that the salt concentration of the loading buffer is compatible with the selected gel. The loading buffer can be diluted if necessary. If the nucleic acid sample is already dissolved in the high-salt buffer, dilute the sample with nuclease-free water before adding the loading buffer. If desired, nucleic acid samples are purified or precipitated and resuspended in nuclease-free water (79) to remove excess salt.
The sample contained a lot of protein	Proteins present in the sample may interfere with the sample mobility in the gel. Proteins were removed by purifying the sample or separated/denatured by preparing the sample in a loading dye loaded with SDS and heating prior to loading.
Loading buffer is not suitable	For electrophoresis of single-stranded nucleic acids, loading dyes containing denaturing agents were used, followed by heating of the sample to prevent the formation of unwanted double-stranded bodies. For electrophoresis of double-stranded DNA, loading dyes containing denaturing agents and heating samples should not be used to protect the double-stranded structure.
Sample loading during the introduction of the gel run
Bubbles	To avoid strip deformation, it should be ensured that bubbles are not trapped in the holes during sample loading.
The loading hole was damaged during loading	During sample loading, the pipette suction tip should be avoided to puncture the sample loading hole.
Sample Wells contain residual acrylamide and/or urea	When using polyacrylamide gels, residual acrylamide (and urea, in the case of denaturing gels) should be rinsed from the Wells before loading.
Too high or too low voltage	Apply voltage as recommended for nucleic acid size range and run buffer used. The voltage is too low or too high to achieve optimal nucleic acid separation.
The electrophoresis time is too short or too long	Run the gel long enough to ensure adequate separation of the bands. However, the electrophoresis time should not be too long, otherwise it will lead to excessive heating, sample denaturation and band diffusion.
The electrophoresis buffer is not suitable	Ensure that the gel preparation matches the electrophoresis buffer and is prepared using the correct method. Use a strong buffer that can sustain electrophoresis for more than 2 hours.
Visualization of examples
Band spread	Do not store gels or leave gels for long periods between completion of electrophoresis and gel color development. Otherwise bands of small molecules and nucleic acid stains in the gel will spread.
Co-migration band	Use appropriate gel percentages, voltages, and running times to separate bands of similar molecular sizes. Co-migration bands usually appear as thick and bright diffuse bands.
The camera out of focus	If viewing the gel through a lens and rendering it on the screen, make sure the camera is in focus.

3 Poorly separated bands

possible reasons	suggestion
gel preparation
Wrong gel ratio	Ensure that the gel percentage is applicable to the sample with the desired content for resolution. High gel percentage is required for small molecule components. In preparation of agarose gels, the volume of the gel should be adjusted by adding water after boiling to compensate for evaporated water and to prevent the percentage of gel from being higher than expected.
The best gel type was not selected	Select the gel type that is more suitable for separating the sample. Polyacrylamide gels are recommended for nucleotide < resolution. 1000 bp.
Poor gel holes formulation	The gel comb should be properly cleaned before gel infusion. To prevent sample leakage from the bottom of the gel, do not push the gel comb all the way to the bottom of the gel. Do not overfill the gel tank, otherwise it will lead to sample hole connection. Allow sufficient time for well formation before removing the gel comb. After the gel has set, remove the gel comb carefully and smoothly to prevent damage to the loading Wells.
Wrong gel type	For electrophoresis of single-stranded nucleic acids, such as RNA, denaturing gels are prepared for efficient separation (124). For electrophoresis of double-stranded DNA, denaturing gels should be avoided to protect the double-stranded structure.
Sample preparation
Sample overload	In gel electrophoresis, do not use excessive samples; A sample dosage of 0.1-0.2 μg per 1 mm gel well width is generally recommended. Trailing, curved or U-shaped bands and fused bands are common manifestations of overload gel.
The sample contained a lot of protein	Proteins present in the sample may interfere with the sample mobility in the gel. Proteins were either removed by purifying the sample or dissociated/denatured by preparing the sample by heating the sample dye and SDS before loading.
Loading buffer is not suitable	For electrophoresis of single-stranded nucleic acids, such as RNA, a loading buffer containing denaturing agent is used, followed by heating of the sample to prevent the formation of undesired double-stranded bodies. For electrophoresis of double-stranded DNA, loading dyes containing denaturing agents and heating samples should not be used to protect the double-stranded structure.
Sample volume is too small	To avoid band bending, ensure that the sample volume is at least 30% of the pore volume.
Gel run
Bubbles are introduced during sample loading	To avoid strip deformation, it should be ensured that bubbles are not trapped in the holes during sample loading.
The loading hole was damaged during loading	During sample loading, the pipette suction tip should be avoided to puncture the sample loading hole.
Sample Wells contain residual acrylamide and/or urea	When using polyacrylamide gels, residual acrylamide (and urea, in the case of denaturing gels) should be rinsed from the Wells before loading.
Too low or too high voltage	Apply voltage for recommended nucleic acid size range and run buffer used. The voltage is too low or too high to achieve optimal nucleic acid separation.
The electrophoresis time is too short or too long	Run the gel long enough to ensure adequate separation of the bands. However, the electrophoresis time should not be too long, otherwise it will lead to excessive heating, sample denaturation and band diffusion.
The electrophoresis buffer is not suitable	Ensure that the gel preparation matches the electrophoresis buffer and is prepared using the correct method. Ensure that the buffer used is appropriate for the sample to be separated. Such as: TAE buffer is more suitable for separating large fragments in the short term (> 1500 bp). TBE for shorter segments (< 5,000 bp) is better, but may slow linear dsDNA migration. Use a strong buffer that can sustain electrophoresis for more than 2 hours.

4 Anomalous separation or migration

possible reasons	suggestion
gel preparation
Uneven gel	When preparing the gel, be sure to mix the matrix solution well. After boiling the agarose gel, there should be no undissolved powder or unmelted solids left.
Uneven gel or inclined loading hole	Place gel tank on flat surface, fill gel and insert comb. The gel comb should be inserted completely perpendicular to the gel surface, parallel to the top edge of the gel, and remain firm as the gel solidifies.
The gel buffer is not suitable	Make sure the gel is prepared in the same buffer as the running buffer.
Gel production
Samples containing different conformations	Different conformations of plasmid DNA (e.g., superhelix, linear, slack/cut) show different mobility in electrophoresis. In the electrophoresis of plasmid DNA, excessive embedding dyes should not be used to avoid changing the conformation of the plasmid. When single-stranded nucleic acids such as RNA are used, a denaturing agent is added to the sample buffer and the sample is heated prior to gel loading to maintain the single strand.
The sample contains a specific sequence	AT - rich DNA migrates more slowly in high-resolution electrophoresis. "Curved" DNA (containing 4-6 adenylate repeats approximately every 10 bp) migrates erratically on polyacrylamide gels. Modified DNA (e.g., methylated, or using biotin or large fluorescent molecular markers) migrates more slowly than unmodified DNA with the same base-pair length.
The sample has a sticky end	Loading buffer containing SDS was used and the sample was heated to prevent nucleic acids with long complementary ends from interacting with each other and forming multiplex.
Proteins bind to nucleic acids	Loading buffer with SDS is used and the sample is heated to limit the interaction between protein and nucleic acid disruption after digestion and ligation, for example.
Gel run
Incompatible run buffer	Ensure that the gel preparation matches the electrophoresis buffer and is prepared using the correct method. Ensure that the buffer used is suitable for the sample separation. Such as: TAE buffer is more suitable for separating large fragments in the short term (1500 bp). TBE for shorter segments (5,000 bp) is better, but may slow linear dsDNA migration. Use a strong buffer that can sustain electrophoresis for more than 2 hours.
Too high voltage	Avoid using voltages that exceed the recommended voltage as it may lead to overheating, sample denaturation and "smiling" bands.
Excessive heating	Ensure that the running buffer has high buffering capacity during long term operation (> 2 hours) during which buffer is circulated/replenished and gel equipment cooled as appropriate. Reduce the voltage, or set the current or power constant.
Visualization of samples
The stain binds to the sample	Consider post-electrophoretic staining when using large fluorescent dyes because the binding of staining to nucleic acids may alter sample mobility during electrophoresis.

5 Incorrect quantitation data

possible reasons	suggestion
gel preparation
Inaccurate molecular weight standards	Ensure that molecular weight standards designed for gel quantification are used. Each band of this class of molecular weight standards contains a known number of fragments.
Different loading dyes were used for samples and molecular weight standards	To obtain reliable quantitative data, the same loading dye should be used for samples and molecular weight standards
Visualization of examples
The band of the selected molecular weight standard is incorrect	The band of interest of the sample was compared with a similar-sized reference band of the quantitative molecular weight standard to improve accuracy.
The strength was not measured properly	To obtain more accurate intensity measurements, the gel background should be subtracted from the measurements of the target band. If available, use the gel imager's built-in quantification software.
Uneven staining	Make sure the fluorescent stain is well mixed with the gel or dye solution. Make sure the gel is completely immersed in the staining solution. For thicker or high percentage gels, the staining time should be extended to ensure full penetration of the fluorescent stain. Alternatively, consider using a stain with faster permeable properties. For denaturing gels, the gel should be cleaned to remove denaturing agents and prevent quenching of fluorescent stains. Alternatively, consider a stain resistant to quench by common denaturants.

6 Other issues

a Sample remains in the gel well

possible reasons	suggestion
gel preparation
Overload samples	In gel electrophoresis, do not use excessive samples; A sample dosage of 0.1-0.2 μg per 1 mm gel well width is generally recommended. Too much sample can result in sample retention in the gel Wells.
Protein and cell remains in the sample	Proteins and cell debris bind to nucleic acids, impeding sample migration. Contaminants were removed by purifying the samples or separating/denaturing them by preparing the samples in a loading dye loaded with SDS and heating them prior to loading.
Gel run
No power	Make sure the power is on/running and connected to the electrophoresis tank. When electrophoresis IS STARTED, CHECK WHETHER THE BUBBLES ON THE immersed ELECTRODE IN the electrophoresis chamber ARE RISING (indicating gas production at the electrode when current flows).
The electrophoresis buffer is not suitable	Ensure that the electrophoresis buffer matches the gel preparation buffer, is prepared correctly, and is conductive.

b Sequence mutations after electrophoresis

possible reasons	suggestion
Visualization of examples
Radiation damage	Try to avoid exposing DNA to UV light. Long wavelength (e.g., 360 nm) UV light rather than short wavelength (e.g., 254 -- 312 nm) UV light should be used for color processing of nucleic acids in gels. Alternatively, using a stain, the DNA is visualized using long excitation wavelengths that are less damaging. In the visualization process, falling illumination is considered instead of transmission illumination.

c Sample floats after loading

possible reasons	suggestion
gel preparation
The buffer was loaded incorrectly	Make sure the loading buffer contains a density component so that the sample sinks into the well.
The sample solution is not suitable	The samples were purified, or the nucleic acid was resuspended in nuclease-free water after precipitation. Residual ethanol or extraction solvents can prevent the sample from sinking to the bottom of the well.

d Speckles in the gel

possible reasons	suggestion
Visualization of examples
Fluorescent contaminant	Certain microorganisms in the dust and/or gel may fluoresce and create spots. Gels are prepared using molecular biology grade reagents and cleaning special laboratory equipment.