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End-point PCR Primers minor

High Molecular Weight Material Stuck in Wells

Symptom
Ethidium bromide-stainable material remains in the gel wells and does not migrate during electrophoresis
Common Causes
  1. 1 Too many PCR cycles performed
  2. 2 Excessive DNA added to reaction
  3. 3 Formation of high molecular weight aggregates or concatenated products
Solutions
  1. 1 Reduce number of PCR cycles
  2. 2 Decrease amount of template DNA added to reaction
  3. 3 Heat sample to 65°C before loading
  4. 4 Place sample on ice immediately after heating and before loading onto gel
Related Video (2)
Bilibili (China-Accessible Mirrors) ★ 78
Step-by-Step PCR Experiment Protocol Guide
"Comprehensive PCR protocol demonstration covering complete workflow and result analysis, essential for understanding how excessive cycles lead to high molecular weight artifacts remaining in wells."
Addgene ★ 75
How to Design Primers for PCR
"Directly addresses PCR primer design, foundational knowledge for understanding how primer quality and quantity affect PCR outcome and potential over-amplification issues."
Source: thermofisher.com ↗
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