Sample Fails to Amplify Despite Positive Control Success (PCR (Sigma Guide))

Question

Accepted Answer

Positive control amplifies successfully but test sample known to contain target shows no amplification; undiluted template fails while dilutions may show improved amplification Common causes: 1) Sample concentration is inappropriate (too high or too low) 2) Inhibition present in the test sample 3) Sample contains PCR inhibitors that are diluted out in lower concentrations Solutions: 1) Test amplification with a diluted sample (1:5 or 1:10 dilution) 2) Add up to 0.3% BSA to the PCR reaction 3) Purify the input nucleic acid to remove inhibitors 4) Spike the reaction with a low level of exogenous target and test for amplification (SPUD Protocol) 5) Perform SPUD assay to confirm inhibition