First-person PCR and gel electrophoresis demonstration

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

Complete Absence of PCR Product

No bands visible on agarose gel after PCR amplification, indicating complete reaction failure.

💡 6 · ✓ 6

critical

No PCR Product Detected

Gel electrophoresis shows no visible band at the expected product size after PCR amplification, indicating complete reaction failure.

💡 8 · ✓ 8

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

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

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

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

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

severe

No Product Due to Suboptimal Cycling Parameters

No amplification product visible despite proper template and reagents, suggesting thermal cycling conditions are inappropriate.

💡 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 Bands or Primer-Dimers Due to Component Imbalance

Multiple bands or primer-dimers appear on gel despite optimized thermal cycling. Component concentrations may be promoting nonspecific primer interactions or amplification.

💡 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

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

Primer Dimer Formation

Small molecular weight band (typically 50-100 bp) visible at bottom of gel, representing primer self-amplification artifacts that compete with target amplification.

💡 3 · ✓ 4

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

Nonspecific Bands or Primer-Dimers

Multiple bands appear on gel in addition to or instead of the expected target band. Primer-dimer artifacts or nonspecific amplification products are visible, indicating lack of reaction specificity.

💡 7 · ✓ 7

moderate

Nonspecific Bands or Primer-Dimers Due to Reagent Issues

Multiple unwanted PCR products appear on gel alongside or instead of target band, caused by reagent concentration imbalances or primer design problems.

💡 4 · ✓ 4

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

moderate

Nonspecific Bands or Primer-Dimers Due to Thermal Cycle Issues

Gel shows multiple bands instead of single target band, or primer-dimers appear as small molecular weight products. The desired product may be present but accompanied by unwanted amplification artifacts.

💡 6 · ✓ 6

moderate

Excessive DNA Smearing on Gel

Amplified DNA appears as broad smear rather than discrete bands, indicating degradation or over-amplification artifacts.

💡 8 · ✓ 8

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