Complete DNA Extraction to Gel Electrophoresis Protocol

critical

No Band Due to Incorrect Component Concentrations

No visible band or very faint band on gel despite proper thermal cycling parameters. The reaction components may be at suboptimal or inhibitory concentrations.

💡 5 · ✓ 5

critical

No Band Due to Omitted Critical Components

Complete absence of PCR product on gel due to missing essential reaction components, representing a critical setup error.

💡 5 · ✓ 5

critical

No DNA Recovered from Gel Extraction or PCR Cleanup

No DNA is recovered after gel extraction or PCR cleanup procedures. Elution yields no measurable nucleic acid.

💡 3 · ✓ 3

severe

No Band or Faint Band Due to Thermal Cycling Errors

No visible band or very faint band appears on the gel after PCR amplification. This indicates insufficient or failed amplification of the target sequence.

💡 6 · ✓ 6

severe

No Band Due to Template or Reagent Quality Issues

PCR fails to produce visible band despite correct concentrations and cycling parameters. Template degradation, contamination, or impure reagents may be inhibiting the reaction.

💡 5 · ✓ 5

severe

No Band or Faint Band Due to Thermal Cycling Issues

After PCR amplification and gel electrophoresis, no visible band appears at the expected product size, or only a very faint band is observed, indicating insufficient or failed amplification.

💡 6 · ✓ 6

severe

No Band or Faint Band Due to Reagent Concentration Issues

Gel shows no amplification product or very weak bands despite correct thermal cycling parameters, indicating problems with reaction component concentrations.

💡 6 · ✓ 6

severe

No Band or Faint Band Due to Suboptimal Thermal Cycling Parameters

No visible PCR product band appears on the gel, or only a very faint band is detected. This occurs despite using appropriate template and primers, suggesting insufficient amplification.

💡 6 · ✓ 6

severe

Low Gel Extraction Yield Due to Improper Gel Dissolution

DNA yield from gel extraction is poor. Undissolved agarose particles may be visible or column flow is impeded.

💡 4 · ✓ 4

severe

Low or No Amplification Due to Template Issues

PCR yields little to no product visible on gel electrophoresis. Expected band is absent or extremely faint despite using standard reaction conditions.

💡 6 · ✓ 6

severe

Sample Fails to Amplify Despite Positive Control Success

Positive control amplifies successfully but test sample known to contain target shows no amplification; undiluted template fails while dilutions may show improved amplification

💡 3 · ✓ 5

severe

PCR Inhibition from Contaminated Template

No or weak PCR amplification despite correct reaction setup, with template DNA containing residual contaminants from extraction or purification that inhibit polymerase activity.

💡 4 · ✓ 6

moderate

No Band or Faint Band Due to Template Quality Problems

PCR fails or produces weak products despite correct reagent concentrations and cycling parameters, due to compromised template DNA quality or presence of inhibitors.

💡 4 · ✓ 4

moderate

Smeared Bands on Gel

PCR products appear as continuous smears rather than discrete bands on gel, indicating template degradation, excessive cycling, or nonspecific amplification throughout a size range.

💡 5 · ✓ 5

moderate

Smeared Bands Due to Template Quality or Concentration Issues

Gel displays smeared bands suggesting degraded products or heterogeneous amplification. Template-related issues such as degradation, contamination, or excessive concentration are suspected.

💡 6 · ✓ 6

moderate

Nonspecific Amplification and Smearing on Gel

Gel shows multiple bands, smears, or high background instead of single clean product band. May include primer-dimers at bottom of gel.

💡 6 · ✓ 6

moderate

Smeared Bands Due to Excessive Thermal Cycling

Gel shows smeared or diffuse bands rather than discrete sharp bands. The smearing pattern suggests heterogeneous product populations from excessive amplification or nonspecific priming.

💡 6 · ✓ 6