Complete DNA Extraction to Gel Electrophoresis Protocol

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

Amplification Failure with Complex Templates

No or weak PCR product when amplifying GC-rich sequences (>65% GC content), long amplicons (>10 kb), or high-complexity genomic DNA despite optimization of standard parameters.

💡 4 · ✓ 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

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

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