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

1. 1 Probe design has significant secondary structure that prevents proper function
2. 2 Probe is inadequately labeled
3. 3 Assay requires optimization

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 (3)

YouTube (Curated Tutorials) ★ 88

The Features Of A Good qPCR Primer Pair

"Directly covers qPCR primer probe design principles, which is the root cause of the probe secondary structure failure."

YouTube (Curated Tutorials) ★ 78

Primer Design: Important Considerations and Tips for Good Primer Design

"Explains important primer/probe design considerations that help avoid secondary structure and assay insensitivity."

Bilibili (China-Accessible Mirrors) ★ 72

qPCR Primer Design: NCBI Screencast Tutorial

"Step-by-step qPCR primer design workflow relevant to preventing probe design defects behind abnormal amplification."