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)

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qPCR Primer Design: NCBI Screencast Tutorial

"Directly addresses qPCR primer/probe design considerations, including avoiding secondary structures."

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The Features Of A Good qPCR Primer Pair

"Explains features of good qPCR primer pairs, crucial for preventing probe secondary structure issues."

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Primer Design: Important Considerations and Tips for Good Primer Design

"General primer design tips that can help avoid secondary structures and improve assay sensitivity."