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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 features, which is critical for understanding probe secondary structure issues that cause the reported assay insensitivity"
Bilibili (China-Accessible Mirrors) ★ 76
qPCR Principles, Experimental Workflow and Results Analysis
"Comprehensive qPCR principles and data interpretation walkthrough provides context for recognizing abnormal amplification plots and baseline drift characteristic of probe design failures"
Source: sigmaaldrich.com ↗
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