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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) ★ 82
Step-by-Step PCR Experiment Protocol Guide
"Hands-on PCR protocol walkthrough shows end-point PCR setup and cycling, giving context for how excessive cycles cause stuck high-MW product."
Addgene ★ 68
How to Design Primers for PCR
"Primer design guidance for PCR is relevant to performing end-point PCR correctly, though it doesn't directly address cycling or gel well artifacts."
Source: thermofisher.com ↗
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