Step-by-Step PCR Experiment Protocol Guide

critical

No PCR Product (Complete Failure)

No bands visible on gel electrophoresis after PCR amplification

💡 10 · ✓ 10

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

Unexpected PCR Product

PCR produces a band of incorrect size or sequence not matching the expected target

💡 5 · ✓ 6

severe

No or Low PCR Amplification Due to Plastic Consumable Issues

PCR reaction fails to produce expected amplification or yields significantly reduced product, despite optimized reagents and cycling parameters. The issue traces to suboptimal thermal transfer or contamination from the plastic vessel itself.

💡 4 · ✓ 4

severe

Plate or Tube Melting and Adhering to Block

After PCR program completion, plates or tubes are found melted or stuck to the thermal cycler block, making removal difficult and potentially damaging samples. Plastic shows signs of thermal degradation.

💡 3 · ✓ 3

severe

PCR Sample Evaporation and Loss During Cycling

Visible reduction in reaction volume after thermal cycling, with condensation on tube caps or film. May result in concentrated reagents, failed reactions, or inability to recover product.

💡 3 · ✓ 3

severe

Low PCR Product Yield

Desired PCR fragment appears faint or barely visible on gel electrophoresis

💡 14 · ✓ 14

severe

GC-Rich Template Amplification Failure

PCR fails to amplify templates with high GC content or produces very low yields

💡 5 · ✓ 8

moderate

PCR Tube Crushing or Deformation Under Lid Pressure

PCR tubes become crushed, collapsed, or deformed after thermal cycling, potentially causing sample loss or compromised seal integrity. Tubes may show visible damage especially when using tube strips.

💡 3 · ✓ 3

moderate

PCR Tube Caps Popping Off During Cycling

Individual tube caps become dislodged during thermal cycling, exposing samples to evaporation and potential contamination. Caps are found loose or completely separated from tubes after run completion.

💡 3 · ✓ 3

moderate

Nonspecific Bands in PCR

Multiple unwanted bands appear on gel electrophoresis alongside or instead of the desired PCR product

💡 4 · ✓ 5

moderate

PCR Product Smearing on Gel

Gel electrophoresis shows smeared or streaked bands instead of distinct sharp bands

💡 6 · ✓ 7

moderate

Primers Stop Working Over Time

Primers that initially worked successfully for PCR gradually fail or produce poor results after storage

💡 4 · ✓ 5

moderate

Pressure-Sensitive Film Not Adhering to Plate

Sealing film peels away from plate wells either before or during thermal cycling, leading to sample evaporation or cross-contamination. Film appears to have poor bonding with well rim surfaces.

💡 2 · ✓ 2

minor

High Molecular Weight Material Stuck in Wells

Ethidium bromide-stainable material remains in the gel wells and does not migrate during electrophoresis

💡 3 · ✓ 4