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.
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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.
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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.
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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.
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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.
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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.
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Sample containing high amounts of protein |
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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.
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Gel run |
Bubbles introduced during sample loading |
- Make sure air bubbles are not trapped in the well during sample loading, to avoid band distortion.
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Well damaged during sample loading |
- Avoid puncturing the wells with the pipette tips during sample loading.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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