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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.
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.
Possible causes for faint bands in gel electrophoresis | Recommendations to avoid or minimize faint bands in gel electrophoresis |
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Low quantity of sample |
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Sample degradation |
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Loading dye masking the desired band |
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Possible causes for faint bands in gel electrophoresis | Recommendations to avoid or minimize faint bands in gel electrophoresis |
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Gel over-run |
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Reversed electrodes |
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Possible causes for faint bands in gel electrophoresis | Recommendations to avoid or minimize faint bands in gel electrophoresis |
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Low sensitivity of stain |
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High background of stain |
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Uneven staining |
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Incorrect light source |
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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.
Possible causes for smearing in gel electrophoresis | Recommendations to minimize smearing in gel electrophoresis |
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Thick gels |
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Poorly formed wells |
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Incorrect gel type |
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Possible causes for smearing in gel electrophoresis | Recommendations to minimize smearing in gel electrophoresis |
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Sample overloading |
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Sample degradation |
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Sample in high-salt buffer |
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Sample containing high amounts of protein |
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Incompatible loading buffer |
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Possible causes for smearing in gel electrophoresis | Recommendations to minimize smearing in gel electrophoresis |
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Bubbles introduced during sample loading |
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Well damaged during sample loading |
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Sample wells containing residual acrylamide and/or urea |
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Very low or high voltage |
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Very short or long run time |
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Incompatible running buffer |
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Possible causes for smearing in gel electrophoresis | Recommendations to minimize smearing in gel electrophoresis |
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Band diffusion |
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Co-migrating bands |
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Out-of-focus camera |
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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.
Possible causes for poor band separation | Recommendations to minimize poor band separation |
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Incorrect gel percentage |
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Suboptimal gel choice |
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Poorly formed wells |
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Incorrect gel type |
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Possible causes for poor band separation | Recommendations to minimize poor band separation |
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Sample overloading |
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Sample containing high amounts of protein |
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Incompatible loading buffer |
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Low volume of sample |
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Possible causes for poor band separation | Recommendations to minimize poor band separation |
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Bubbles introduced during sample loading |
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Well damaged during sample loading |
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Sample wells containing residual acrylamide and/or urea |
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Very low or high voltage |
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Very short or long run time |
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Incompatible running buffer |
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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.
Possible causes for anomalous band separation | Recommendations to minimize anomalous migration |
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Nonhomogeneous gel concentration |
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Uneven gels or slanted wells |
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Incorrect gel buffer |
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Possible causes of anomalous migration | Recommendations to minimize anomalous migration |
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Sample containing different conformations |
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Sample of unusual sequences |
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Sample with cohesive ends |
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Proteins bound to nucleic acids |
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Possible causes of anomalous migration | Recommendations to minimize anomalous migration |
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Incompatible running buffer |
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Very high voltage |
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Excessive heat generation |
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Possible causes of anomalous migration | Recommendations to minimize anomalous migration |
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Stain binding to sample |
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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.
Possible causes for incorrect quantitation data | Recommendations to help minimize incorrect quantitation data |
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Incorrect ladder |
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Different loading dyes used for sample and ladder |
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Possible causes for incorrect quantitation data | Recommendations to minimize incorrect quantitation data |
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Incorrect band of the ladder chosen |
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Improper intensity measurement |
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Uneven staining |
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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.
Possible causes for sample to remain in gel wells | Recommendations to improve sample migration |
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Sample preparation | |
Overloaded sample |
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Protein and cell debris in the sample |
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Gel run | |
No power |
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Incorrect running buffer |
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Possible causes of sequence mutations after electrophoresis | Recommendations to prevent sequence mutations after electrophoresis |
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Radiation damage |
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Possible causes of sample floatation | Recommendations to prevent sample floatation |
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Incorrect loading buffer |
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Sample in incompatible solution |
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Possible causes for speckles in gel | Recommendations to minimize speckles in the gel |
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Fluorescing contaminants |
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For Research Use Only. Not for use in diagnostic procedures.