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Troubleshooting Nucleic Acid Gels

Dec 4, 2025

Overview

  • Topic: Troubleshooting nucleic acid gel electrophoresis (common problems and solutions).
  • Purpose: Identify causes and practical recommendations to fix faint bands, smearing, poor separation, anomalous migration, incorrect quantitation, and other issues.
  • Scope: Gel preparation, sample preparation, gel run conditions, and visualization recommendations.

Faint Bands

  • Description: Bands appear weak, fuzzy, or absent; hard to visualize.
  • Main causes: Low sample quantity, sample degradation, loading dye masking bands, gel over-run, reversed electrodes, low stain sensitivity, high background, uneven staining, incorrect light source.
Cause CategorySpecific CauseRecommended Actions
Gel preparationLow sample quantityLoad ≥0.1–0.2 µg DNA/RNA per mm of well width; use deep narrow wells.
Gel preparationSample degradationUse molecular-biology-grade reagents, nuclease-free labware, wear gloves.
Gel preparationLoading dye masking bandCheck dye migration; use different dye or adjust sample volume.
Gel runGel over-runMonitor run time and dye migration; avoid running small fragments off gel.
Gel runReversed electrodesEnsure wells are toward negative electrode for horizontal gels.
VisualizationLow stain sensitivityUse more stain, longer staining, or higher-affinity stain for ss nucleic acids.
VisualizationHigh stain backgroundDestain or choose low-intrinsic-fluorescence stain.
VisualizationUneven stainingMix in-gel stain thoroughly; fully submerge gel for post-staining with gentle shaking.
VisualizationIncorrect light sourceUse correct excitation wavelength for the fluorescent dye.

Smeared Bands

  • Description: Diffuse, blurry bands overlapping neighbors; poor resolution.
  • Main causes: Thick gels, poorly formed wells, incorrect gel type, sample overloading, degradation, high-salt buffer, protein contamination, incompatible loading buffer, bubbles while loading, damaged wells, wrong voltage or run time, incompatible running buffer, band diffusion, co-migration, out-of-focus imaging.
Cause CategorySpecific CauseRecommended Actions
Gel prepThick gelsCast agarose 3–4 mm thick; avoid >5 mm thickness.
Gel prepPoorly formed wellsUse clean combs; do not push comb to bottom; avoid overfilling tray.
Gel prepIncorrect gel typeUse denaturing gels for ss nucleic acids; avoid denaturing for dsDNA.
Sample prepOverloadingLimit to 0.1–0.2 µg per mm of well width.
Sample prepDegradationUse nuclease-free conditions and good lab practices.
Sample prepHigh-salt bufferDilute or purify sample to remove excess salt.
Sample prepProtein contaminationPurify or denature proteins (SDS + heat) before loading.
Sample prepIncompatible loading bufferUse denaturant-containing dye and heat for ss samples; avoid for dsDNA.
Gel runBubbles/damaged wellsAvoid trapped air; do not puncture wells with tips.
Gel runVoltage/run timeUse recommended voltage and appropriate run time; avoid extremes.
Gel runIncompatible bufferPrepare gel and running buffers correctly and compatibly.
VisualizationBand diffusionVisualize soon after run; avoid long gel storage.
VisualizationCo-migrating bandsAdjust gel percentage, voltage, and run time to resolve similar sizes.
VisualizationImaging issuesEnsure camera in focus.

Poorly Separated Bands

  • Description: Bands closely stacked and not clearly differentiated.
  • Main causes: Incorrect gel percentage or type, poorly formed wells, sample overloading, protein contamination, incompatible loading buffer, low sample volume, bubbles/damaged wells, incorrect voltage/run time, incompatible running buffer.
Cause CategorySpecific CauseRecommended Actions
Gel prepIncorrect gel percentageChoose gel percentage appropriate for target fragment sizes; compensate for evaporative concentration.
Gel prepSuboptimal gel choiceUse polyacrylamide for fragments <1,000 bp; choose gel type for resolution needs.
Gel prepPoorly formed wellsClean combs; avoid pushing comb to bottom; remove comb carefully.
Sample prepOverloadingLimit 0.1–0.2 µg per mm well width.
Sample prepProtein contaminationPurify or denature proteins (SDS + heat) before loading.
Sample prepIncompatible loading bufferUse denaturant and heat for ss nucleic acids; avoid denaturant for dsDNA.
Sample prepLow volumeFill at least 30% of well to avoid distortion.
Gel runBubbles/damaged wellsPrevent air traps; avoid puncturing wells.
Gel runVoltage/run timeUse recommended voltage and run time for size range; avoid overheating or under-running.
Gel runIncompatible bufferSelect TAE for large fragments (>1,500 bp) in short runs; TBE for smaller fragments.

Anomalous Separation or Migration (Smiling Bands)

  • Description: Irregular migration patterns (e.g., smiling) caused by uneven heat or electric field.
  • Main causes: Nonhomogeneous gel concentration, uneven or slanted wells, incorrect gel buffer, sample conformations, unusual sequences, cohesive ends, protein-bound nucleic acids, incompatible running buffer, high voltage, excessive heat, stain-binding shifts.
Cause CategorySpecific CauseRecommended Actions
Gel prepNonhomogeneous concentrationMix matrix thoroughly; ensure agarose fully dissolved.
Gel prepUneven/slanted wellsLevel tray; insert comb vertical and stable.
Gel prepIncorrect gel bufferPrepare gel in same buffer as running buffer.
Sample prepDifferent conformationsRecognize plasmid forms; limit intercalating dye and avoid excessive dye.
Sample prepUnusual sequences/modificationsExpect altered mobility for AT-rich, curved, methylated, or labeled DNA.
Sample prepCohesive endsUse loading buffer with SDS and heat to prevent concatemer formation.
Sample prepProtein bindingDisrupt with SDS-containing loading buffer and heat.
Gel runIncompatible running bufferUse compatible buffers; choose TAE or TBE per fragment size and runtime.
Gel runVery high voltageAvoid exceeding recommended voltage to limit heat and smiling.
Gel runExcessive heat generationImprove cooling, replenish/ circulate buffer, lower voltage or control current.
VisualizationStain binding shiftConsider post-electrophoresis staining to avoid mobility shifts from large stains.

Incorrect Quantitation

  • Description: Wrong concentration estimates due to ladder or dye mismatch and uneven staining.
  • Main causes: Incorrect ladder type, different loading dyes, wrong ladder band chosen, improper intensity measurement, uneven staining.
Cause CategorySpecific CauseRecommended Actions
Sample prepIncorrect ladderUse ladders designed for quantitation (known band quantities).
Sample prepDifferent loading dyesUse the same loading dye for sample and ladder.
VisualizationWrong ladder band chosenCompare sample to similarly sized reference band on quantitative ladder.
VisualizationImproper intensity measurementSubtract gel background; use gel imager quantitation software.
VisualizationUneven stainingEnsure stain homogenization, full submersion, adequate staining time; consider stains with better penetration or resistance to quenching.

Other Common Problems

  • Samples remaining in wells:
    • Causes: Overloading, protein/cell debris, no power, incorrect running buffer.
    • Actions: Reduce load, purify sample or denature contaminants, verify power supply and bubbling at electrodes, ensure compatible conductive buffer.
  • Sample floatation:
    • Causes: Loading buffer lacks density agent, sample in incompatible solution or solvent carryover.
    • Actions: Use loading dye with density ingredient; purify and resuspend sample in nuclease-free water to remove ethanol/solvent.
  • Speckles in gel:
    • Causes: Fluorescing contaminants like dust or microorganisms.
    • Actions: Use molecular-biology-grade reagents and clean dedicated labware.
  • Preventing sequence mutations after electrophoresis:
    • Cause: UV radiation damage.
    • Actions: Minimize UV exposure; use longer-wavelength UV (e.g., 360 nm), stains with long excitation wavelengths, or epi-illumination instead of transillumination.

Key Terms and Definitions

  • Loading dye: Solution added to samples to increase density and track migration; may affect visibility if similar migration size.
  • Denaturing gel: Gel containing denaturant to keep single-stranded nucleic acids unfolded (used for RNA).
  • TAE/TBE: Common running buffers; TAE better for large fragments in short runs, TBE for smaller fragments but can slow migration.
  • Quantitative ladder: DNA ladder with bands of known quantity to enable gel-based quantitation.

Action Items / Next Steps (Practical Checklist)

  • Before casting gel: confirm buffer selection (TAE vs TBE), calculate gel percentage, use clean comb and level tray.
  • Before loading samples: measure and dilute/purify samples to appropriate concentration and salt content; add proper loading dye and fill ≥30% of well.
  • During run: set recommended voltage, monitor dye front and electrode bubbles, cool or circulate buffer for long runs.
  • After run: minimize time before visualization, choose appropriate staining method (post-stain if stain shifts mobility), use correct light source, and quantify using a quantitative ladder and background subtraction.
  • If persistent problems: systematically vary one parameter at a time (gel percentage, voltage, buffer, staining) to identify root cause.

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