Abnormal melt curve (multiple peaks or shoulder) (qPCR (RT-qPCR))

Question

Accepted Answer

Melt curve shows multiple peaks or a shoulder, Tm does not match expectation, gel shows multiple bands. Common causes: 1) Primer dimers (low Tm peak around 65 – 75 °C) 2) Non-specific amplification products 3) Primer design is suboptimal 4) Mg²⁺ concentration too high promotes non-specific binding 5) Annealing temperature too low Solutions: 1) Re-design primers (check Tm match, avoid 3' complementarity) 2) Raise annealing temperature 1 – 3 °C 3) Optimize Mg²⁺ concentration 4) Lower primer concentration 5) Use a hot-start polymerase to reduce non-specific amplification before cycling begins