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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)
YouTube (Curated Tutorials) ★ 82
Primer Design: Important Considerations and Tips for Good Primer Design
"Covers primer design considerations that directly govern annealing temperature and specificity, the root cause of wrong-sized products."
Addgene ★ 74
Polymerase Chain Reaction (PCR) Protocol
"Walks through the PCR protocol including cycling parameters, letting viewers see where annealing temperature is set and how to perform PCR correctly."
Bilibili (China-Accessible Mirrors) ★ 71
First-person PCR and gel electrophoresis demonstration
"Combines PCR workflow with gel electrophoresis, matching the symptom observation and the technique context where this failure is detected."
Source: neb.com ↗
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