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PCR (Invitrogen Guide) severe

Amplification Failure from Primer Problems

Symptom
No visible PCR product despite good template quality. Control reactions with different primers may work normally.
Common Causes
  1. 1 Primers not specific to target or complementary to wrong strand
  2. 2 Degraded or old primers with reduced binding efficiency
  3. 3 Primer concentration insufficient (below 0.1 μM range)
  4. 4 Primer design issues preventing proper target binding
Solutions
  1. 1 Verify primers are specific to target and complementary to correct strands using online design tools
  2. 2 Aliquot primers after resuspension; reconstitute fresh aliquots or obtain new primers
  3. 3 Optimize primer concentrations to 0.1–1 μM (minimum 0.5 μM for long PCR or degenerate primers)
  4. 4 Review and redesign primers ensuring proper Tm and target specificity
Related Video (3)
Addgene ★ 90
How to Design Primers for PCR
"Directly addresses primer design for PCR, the exact root cause of this failure."
YouTube (Curated Tutorials) ★ 85
Primer Design: Important Considerations and Tips for Good Primer Design
"Covers key primer design considerations and tips that prevent nonspecific or wrong-strand primers."
Addgene ★ 60
Polymerase Chain Reaction (PCR) Protocol
"Walks through the PCR protocol itself, giving context for where amplification fails with bad primers."
Source: thermofisher.com ↗
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