Abnormal Amplification Plots Due to Excessive Template Concentration

Symptom

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

1. 1 Too much template included in the PCR reaction
2. 2 Template concentration is too high for the reaction to be efficient
3. 3 Instrument cannot effectively process baseline data at very high template concentrations

1. 1 Dilute template to appropriate working concentration
2. 2 Use template quantity within recommended range for the protocol
3. 3 Prepare fresh dilution series with appropriate concentrations
4. 4 Verify template quantification accuracy