Irregular Standard Curve Spacing Due to Sample Inhibitors (PCR (Sigma Guide))

Question

Accepted Answer

Cq data for standard curve dilutions are irregularly spaced; ΔCq between dilutions is inconsistent and decreases with increasing dilutions; replicates are precise but pattern is abnormal Common causes: 1) Sample contains an inhibitor that affects concentrated samples more than diluted ones 2) Errors in the dilutions of the template for the standard curve 3) Inappropriate assay design leading to inefficient priming Solutions: 1) Add up to 0.3% BSA to the PCR reaction 2) Purify the input nucleic acid to remove inhibitors 3) Test amplification with diluted sample (1:5 or 1:10 dilution series) 4) Run SPUD assay to identify samples containing inhibitors 5) Repeat assay on freshly prepared serial dilution to rule out pipetting errors