Insensitive Assay with Abnormal Amplification Due to Probe Secondary Structure (PCR (Sigma Guide))

Question

Accepted Answer

Assay is insensitive and amplification plots look abnormal with pronounced drift of the baseline; fluorescence signals are weak or irregular Common causes: 1) Probe design has significant secondary structure that prevents proper function 2) Probe is inadequately labeled 3) Assay requires optimization Solutions: 1) Re-design the assay if possible to avoid probe secondary structure 2) When redesign is not possible, add <10% final reaction volume of betaine or DMSO to reaction buffers 3) Further optimize reaction conditions 4) Request design review by technical support team