Nucleic acid electrophoresis is a critical step in many molecular biology applications. Problems during electrophoresis can affect downstream experiments and reduce overall workflow efficiency. This section discusses common issues in nucleic acid gel electrophoresis and recommended solutions. For agarose-specific casting and grade selection, see how gel electrophoresis works and how to choose agarose powder, or browse the Agarose (CAS 9012-36-6) product page.
Topics covered:
- No bands or barely visible bands
- Smeared or diffuse bands
- Poor band separation
- Abnormal band separation or migration
- Incorrect quantification data
- Other issues (sample retained in wells; sequence mutations after electrophoresis; floating samples after loading; spots in the gel)
1. No bands or barely visible bands
| Possible cause | Suggested solutions |
| Gel preparation | • Ensure sufficient sample is loaded. Typically, several nanograms per band are needed for visibility; a common recommendation is 0.1–0.2 μg sample per 1 mm of well width.
• Use a comb with deep wells to improve visualization of small sample amounts. |
| Sample degradation | • Use molecular-biology-grade reagents and nuclease-free labware. Follow good laboratory practice when handling nucleic acids (e.g., wear gloves, prevent nuclease contamination, work in designated areas)—especially for RNA. |
| Loading dye masking the target band | • Check the migration position of the loading dye. Dyes can mask and hide target bands, especially when sample amounts are low. |
| Gel over-run | • Monitor run time and dye migration to avoid smaller sample molecules running off the gel. |
| Electrodes reversed | • Ensure electrodes are correctly connected to the power supply. For horizontal gels, wells should be on the same side as the negative electrode (cathode). |
| Low staining sensitivity | • Check the manufacturer’s stated sensitivity for the fluorescent dye used to detect nucleic acids.
• Increase dye amount or staining time to visualize single-stranded nucleic acids; or use dyes with higher affinity for ss molecules to improve specificity and sensitivity. • For thicker or high-percentage agarose gels, extend staining time for adequate dye penetration, or use dyes with faster penetration. |
| High dye background | • Destain the gel, or choose a dye with low intrinsic fluorescence to skip destaining. |
| Uneven staining | • If samples are only partially visible or visible only in some lanes:
• a. For in-gel staining: mix the stain thoroughly into the agarose solution during gel casting (avoid bubbles). • b. For post-electrophoresis staining: fully submerge the gel in stain and gently shake for an adequate time. |
| Incorrect light source | • If using a fluorescent dye, check its excitation wavelength and ensure the light source is suitable for visualization. |
2. Smeared or diffuse bands
| Possible cause | Suggested solutions |
| Gel preparation / thickness | • When casting horizontal agarose gels, keep gel thickness at 3–4 mm. Thickness above 5 mm may cause band diffusion during electrophoresis. |
| Poor well formation | • Clean the comb properly before casting.
• Do not push the comb all the way to the bottom of a horizontal gel, to prevent leakage from the well bottom and band smearing. • Do not overfill the gel tray, which can cause wells to connect. • Allow enough time for wells to form before removing the comb. • After the gel solidifies, remove the comb carefully and steadily to avoid damaging wells. |
| Wrong gel type | • For single-stranded nucleic acids (e.g., RNA), prepare a denaturing gel for effective separation; for double-stranded DNA, avoid denaturing gels. |
| Sample overload | • Do not overload; typically recommend 0.1–0.2 μg sample per 1 mm well width. Smearing, bent or U-shaped bands, and fused bands are common signs of overload. |
| Sample degradation | • Use molecular-biology-grade reagents and nuclease-free labware; follow good practice when handling nucleic acids, especially RNA. |
| Sample in high-salt buffer | • Check whether loading-buffer salt concentration is compatible with the selected gel; dilute if needed.
• If the nucleic acid is already in high-salt buffer, dilute with nuclease-free water before adding loading buffer. If necessary, purify or precipitate and resuspend in nuclease-free water to remove excess salt. |
| High protein content | • Proteins in the sample may interfere with migration. Purify to remove protein, or prepare samples in SDS-containing loading dye and heat before loading to dissociate/denature proteins. |
| Inappropriate loading buffer | • For ss nucleic acids: use loading dye containing denaturant, then heat the sample to prevent unwanted duplexes.
• For dsDNA: do not use denaturant-containing loading dye or heat the sample, to preserve double-stranded structure. |
| Air bubbles | • During loading, ensure bubbles are not trapped in wells to avoid band distortion. |
| Wells damaged during loading | • Avoid piercing wells with pipette tips while loading. |
| Residual acrylamide or urea in wells | • For polyacrylamide gels, rinse wells before loading to remove residual acrylamide (and urea for denaturing gels). |
| Voltage too low or too high | • Apply voltage according to the recommended range for nucleic acid size and running buffer. Extremes of voltage prevent optimal separation. |
| Run time too short or too long | • Run long enough for adequate band separation, but not so long that overheating, sample denaturation, and band diffusion occur. |
| Inappropriate electrophoresis buffer | • Ensure gel casting buffer matches the running buffer and is prepared correctly.
• Use a strong buffer that can support runs longer than 2 hours. |
| Band diffusion after the run | • Do not store the gel for long periods between electrophoresis and imaging; small molecules and nucleic-acid dyes will diffuse. |
| Co-migrating bands | • Use appropriate gel percentage, voltage, and run time to separate similarly sized bands. Co-migrating bands often appear as thick, bright, diffuse bands. |
| Camera out of focus | • If imaging through a camera lens onto a screen, ensure the camera is focused. |
3. Poor band separation
| Possible cause | Suggested solutions |
| Unsuitable gel concentration | • Ensure gel concentration is appropriate for resolving the desired sample content; small molecules require higher gel concentration.
• When preparing agarose gels, after boiling add water to adjust volume to compensate for evaporation and avoid higher-than-intended concentration. |
| Unsuitable gel type | • Choose a gel type better suited to the sample. Polyacrylamide gels are recommended for resolving nucleotides <1,000 bp. |
| Poor wells | • Clean the comb before casting; do not push the comb to the gel bottom; do not overfill the tray; allow wells to form fully; remove the comb carefully after solidification. |
| Wrong gel type (denaturing vs native) | • For ss nucleic acids (e.g., RNA), prepare a denaturing gel for effective separation.
• For dsDNA, avoid denaturing gels to preserve double-stranded structure. |
| Sample overload | • Do not overload; typically 0.1–0.2 μg per 1 mm well width. Smearing, bent/U-shaped, and fused bands indicate overload. |
| High protein content | • Purify to remove protein, or prepare samples with SDS-containing loading dye and heat before loading to dissociate/denature proteins. |
| Inappropriate loading buffer | • For ss nucleic acids (e.g., RNA): use denaturant-containing loading buffer, then heat to prevent unwanted duplexes.
• For dsDNA: do not use denaturant-containing loading dye or heat the sample. |
| Sample volume too low | • To avoid bent bands, ensure sample volume is at least 30% of well volume. |
| Bubbles introduced during loading | • Ensure bubbles are not trapped in wells during loading. |
| Wells damaged during loading | • Avoid piercing wells with pipette tips. |
| Residual acrylamide or urea in wells | • Rinse polyacrylamide wells before loading to remove residual acrylamide (and urea for denaturing gels). |
| Voltage too low or too high | • Apply the recommended voltage for nucleic acid size range and running buffer. |
| Run time too short or too long | • Run long enough for separation, but avoid excessive run time that causes overheating, denaturation, and diffusion. |
| Inappropriate electrophoresis buffer | • Match gel and running buffers; prepare them correctly.
• Choose a buffer suited to the sample. For example: (a) TAE is better for large fragments (>1,500 bp) in shorter runs; (b) TBE is better for shorter fragments (<5,000 bp) but may slow linear dsDNA migration. • Use a high-buffering-capacity buffer that can support runs longer than 2 hours. |
4. Abnormal band separation or migration
| Possible cause | Suggested solutions |
| Uneven gel concentration | • Mix the matrix solution thoroughly when preparing the gel. After boiling agarose, no undissolved powder or unmelted solids should remain. |
| Uneven gel or tilted wells | • Place the gel tray on a flat surface when casting and inserting the comb. Insert the comb fully vertical, parallel to the top edge of the gel, and keep it stable while the gel sets. |
| Inappropriate gel buffer | • Prepare the gel in the same buffer as the running buffer. |
| Sample contains different conformations | • Different plasmid DNA conformations (e.g., supercoiled, linear, relaxed/nicked) migrate differently.
• Do not use excessive intercalating dye during plasmid electrophoresis, which can alter plasmid conformation. • For ss nucleic acids such as RNA, add denaturant to the sample buffer and heat before loading to keep molecules single-stranded. |
| Sample contains special sequences | • AT-rich DNA migrates more slowly in high-resolution electrophoresis.
• “Bent” DNA (about 4–6 adenosine repeats every 10 bp) migrates anomalously on polyacrylamide gels. • Modified DNA (e.g., methylated, or labeled with biotin or large fluorophores) migrates more slowly than unmodified DNA of the same base-pair length. |
| Sticky ends on the sample | • Use SDS-containing loading buffer and heat the sample to prevent molecules with long complementary ends from interacting and forming concatemers. |
| Protein bound to nucleic acid | • Disrupt protein–nucleic acid interactions—e.g., after restriction digestion and ligation, use SDS-containing loading buffer and heat the sample. |
| Incompatible electrophoresis buffer | • Match gel and running buffers; prepare correctly.
• Use a buffer suited to the sample: TAE for large fragments (>1,500 bp) in shorter runs; TBE for shorter fragments (<5,000 bp), noting it may slow linear dsDNA. • Use a high-buffering-capacity buffer for runs >2 hours. |
| Voltage too high | • Avoid voltages above the recommended range; excess voltage can cause overheating, sample denaturation, and “smiling” bands. |
| Excessive heating | • Ensure high buffering capacity; circulate/replenish buffer during long runs (>2 hours); cool the gel apparatus when appropriate.
• Reduce voltage, or set constant current or constant power. |
| Stain binding to sample during the run | • When using large fluorescent dyes, consider post-electrophoresis staining, because dye binding during electrophoresis can alter migration. |
5. Incorrect quantification data
| Possible cause | Suggested solutions |
| Inaccurate molecular-weight standard | • Use a molecular-weight standard designed specifically for gel quantification. |
| Different loading dyes for sample and standard | • For reliable quantification, use the same loading dye for samples and molecular-weight standards. |
| Incorrect reference band in the standard | • Compare bands of interest with similarly sized reference bands in the quantitative molecular-weight standard to improve accuracy. |
| Inappropriate intensity measurement | • For more accurate intensity measurement, subtract gel background from the target-band measurement. If available, use the built-in gel electrophoresis image analysis software of the gel imager. |
| Uneven staining | • Ensure fluorescent stain is thoroughly mixed with the gel or staining solution.
• Ensure the gel is fully submerged in stain. • For thicker or high-percentage gels, extend staining time for adequate penetration, or use faster-penetrating dyes. • For denaturing gels, wash to remove denaturants that quench fluorescent stains, or use a dye resistant to quenching by common denaturants. |
6. Other gel electrophoresis issues
6a. Sample retained in the wells
| Possible cause | Suggested solutions |
| Sample overload | • Do not overload; typically 0.1–0.2 μg per 1 mm well width. Excess sample can remain trapped in the wells. |
| Protein and cell debris in the sample | • Protein and cell debris bound to nucleic acids can hinder migration. Purify to remove contaminants, or prepare samples in SDS-containing loading dye and heat before loading to dissociate/denature them. |
| No power / no current | • Confirm the power supply is on/running and connected to the tank.
• When electrophoresis starts, check for bubbles rising from submerged electrodes (indicating gas evolution when current flows). |
| Inappropriate electrophoresis buffer | • Ensure running buffer matches gel casting buffer, is prepared correctly, and is conductive. |
6b. Sequence mutations after electrophoresis
| Possible cause | Suggested solutions |
| Radiation / UV damage | • Minimize DNA exposure to UV light.
• When visualizing nucleic acids in gels, use long-wavelength UV (e.g., 360 nm) rather than short-wavelength UV (e.g., 254–312 nm). • Alternatively, use dyes with longer excitation wavelengths that cause less sample damage. • Consider epi-illumination rather than transillumination during visualization. |
6c. Sample floats after loading
| Possible cause | Suggested solutions |
| Incorrect loading buffer | • Ensure the loading buffer contains a density agent so the sample sinks into the well. |
| Inappropriate sample solution | • Purify the sample, or precipitate and resuspend nucleic acids in nuclease-free water. Residual ethanol or extraction solvents can prevent the sample from sinking. |
6d. Spots in the gel
| Possible cause | Suggested solutions |
| Fluorescent contaminants | • Dust or certain microorganisms in the gel may fluoresce and appear as spots. Prepare gels with molecular-biology-grade reagents and clean, dedicated labware. |
For additional troubleshooting advice, contact Monuo Chemical. You may also find our agarose troubleshooting notes useful: smeared bands, weak signals, and soft gels, or request a quote for electrophoresis-grade agarose powder.
Related reading: agarose applications · introduction to agarose · contact us.