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PCR (Sigma Guide) severe

Insensitive Assay with Abnormal Amplification Due to Probe Secondary Structure

Symptom
Assay is insensitive and amplification plots look abnormal with pronounced drift of the baseline; fluorescence signals are weak or irregular
Common Causes
  1. 1 Probe design has significant secondary structure that prevents proper function
  2. 2 Probe is inadequately labeled
  3. 3 Assay requires optimization
Solutions
  1. 1 Re-design the assay if possible to avoid probe secondary structure
  2. 2 When redesign is not possible, add <10% final reaction volume of betaine or DMSO to reaction buffers
  3. 3 Further optimize reaction conditions
  4. 4 Request design review by technical support team
Related Video (2)
YouTube (Curated Tutorials) ★ 82
The Features Of A Good qPCR Primer Pair
"Directly addresses qPCR primer pair design quality, which is essential for understanding how probe/primer secondary structure failures manifest in real-time PCR assays."
Bilibili (China-Accessible Mirrors) ★ 78
qPCR Principles, Experimental Workflow and Results Analysis
"Comprehensive qPCR principles and data interpretation walkthrough that would help researchers recognize abnormal amplification plots and baseline drift characteristic of this failure mode."
Source: sigmaaldrich.com ↗
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