PCR protocol fundamentals—hands-on operation guide

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 or Faint Band Due to Incorrect Component Concentrations

PCR amplification fails or produces very weak bands. Thermal cycling parameters appear correct, but reaction components may be improperly balanced or missing.

💡 6 · ✓ 6

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

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

Insufficient Amplification from Polymerase Issues

Weak or absent PCR product. Primers are degraded or show primer-dimer formation at the bottom of gel.

💡 4 · ✓ 5

severe

Sequence Errors in PCR Product

Sequencing of the PCR product reveals mutations, insertions, deletions, or other sequence variations compared to the original template DNA.

💡 6 · ✓ 6

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

PCR污染和携带污染

Unexpected products appear in negative controls or multiple samples show identical non-specific bands, indicating contamination from previous amplifications or environmental sources.

💡 4 · ✓ 7

moderate

Abnormal Amplification Plots Due to Excessive Template Concentration

Very low Cq values for concentrated samples; amplification plots are not regularly spaced and appear abnormal; background fluorescence is significantly higher; minimal fluorescence yield through the reaction

💡 3 · ✓ 4

moderate

Amplification Failure from Suboptimal Cycling Parameters

No product or very weak bands despite proper template and primer quality. Reaction components appear functional in other assays.

💡 4 · ✓ 4