Complete DNA Extraction to Gel Electrophoresis Protocol

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

Incorrect PCR Product Size

Gel electrophoresis shows PCR product band(s) at unexpected molecular weight, either larger or smaller than the predicted amplicon size.

💡 3 · ✓ 4

severe

Multiple or Non-Specific PCR Products

Gel electrophoresis reveals multiple bands, smearing, or bands at incorrect sizes in addition to or instead of the expected product, indicating lack of amplification specificity.

💡 7 · ✓ 7

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

severe

No Band or Faint Band with GC-Rich Templates

PCR fails or produces very weak amplification specifically with high GC content templates (>65%). Standard protocols work with other templates but not GC-rich sequences.

💡 3 · ✓ 4

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 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

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