qPCR Primer Design: NCBI Screencast Tutorial

severe

Insensitive Assay with Abnormal Amplification Due to Probe Secondary Structure

Assay is insensitive and amplification plots look abnormal with pronounced drift of the baseline; fluorescence signals are weak or irregular

💡 3 · ✓ 4

severe

PCR Efficiency Greater Than 120% with Inconsistent ΔCq

PCR efficiency calculated from standard curve is greater than 120%; ΔCq between 10-fold dilutions is much less than expected 3.3 cycles (e.g., 1.5 cycles); standard curve gradient indicates abnormally high efficiency

💡 4 · ✓ 6

severe

Abnormal melt curve (multiple peaks or shoulder)

Melt curve shows multiple peaks or a shoulder, Tm does not match expectation, gel shows multiple bands.

💡 5 · ✓ 5

severe

No DNA Amplification in ChIP Samples

No PCR product is detected in ChIP samples by qPCR or gel electrophoresis. Ct values fail to appear or remain above threshold, and no visible bands are observed on gels.

💡 5 · ✓ 6

moderate

Nonspecific Amplification or Multiple Melt Peaks in SYBR Green qPCR

Multiple products or melt curve peaks appear in SYBR Green qPCR after ChIP, indicating amplification of off-target sequences. Gel electrophoresis may reveal bands at incorrect sizes or multiple bands.

💡 4 · ✓ 5

moderate

Sequence Errors at PCR Product Termini

Sequencing shows errors, truncations, or unexpected sequences specifically at the 5′ or 3′ ends of amplified products.

💡 4 · ✓ 5