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PCR (Polymerase Chain Reaction) severe

Incorrect PCR Product Size

Symptom
Gel electrophoresis shows PCR product band(s) at unexpected molecular weight, either larger or smaller than the predicted amplicon size.
Common Causes
  1. 1 Incorrect annealing temperature causing non-specific primer binding
  2. 2 Mispriming due to primers having complementary regions within template DNA
  3. 3 Suboptimal Mg²⁺ concentration affecting polymerase processivity and specificity
Solutions
  1. 1 Recalculate primer Tm values using NEB Tm calculator and adjust annealing temperature accordingly
  2. 2 Verify primers have no additional complementary regions within template DNA using BLAST or primer design software
  3. 3 Optimize Mg²⁺ concentration by testing increments of 0.2–1 mM
  4. 4 Repeat reactions using fresh reagent solutions to eliminate nuclease contamination
Related Video (3)
Addgene ★ 80
Polymerase Chain Reaction (PCR) Protocol
"Walks through the standard PCR protocol including annealing steps, directly relevant to performing the technique correctly and avoiding the incorrect annealing temperature that causes wrong product si"
Bilibili (China-Accessible Mirrors) ★ 75
First-person PCR and gel electrophoresis demonstration
"First-person PCR plus gel electrophoresis demonstration shows exactly the equipment and workflow where unexpected band sizes would be observed and diagnosed."
YouTube (Curated Tutorials) ★ 70
Primer Design: Important Considerations and Tips for Good Primer Design
"Covers primer design considerations that affect specificity and annealing, relevant context for understanding why non-specific primer binding yields incorrect product sizes."
Source: neb.com ↗
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