Abnormal Amplification Plots Due to Excessive Template Concentration (PCR (Sigma Guide))

Question

Accepted Answer

Very low Cq values for concentrated samples; amplification plots are not regularly spaced and appear abnormal; background fluorescence is significantly higher; minimal fluorescence yield through the reaction Common causes: 1) Too much template included in the PCR reaction 2) Template concentration is too high for the reaction to be efficient 3) Instrument cannot effectively process baseline data at very high template concentrations Solutions: 1) Dilute template to appropriate working concentration 2) Use template quantity within recommended range for the protocol 3) Prepare fresh dilution series with appropriate concentrations 4) Verify template quantification accuracy