QPCR Troubleshooting Guide
| Problem | Possible solution |
| Low or no fluorescence or high Ct | If you use a probe, it may be incompatible, poorly synthesized, or degraded. Try using different probes. • Verify that the fluorescence data is collected in the correct step • Gradually increase the primer annealing time • Gradually lower the primer application temperature • Gradually increase the elongation time • The amplicon size should not exceed 250 bp • Increase the amount of template • Make sure the template sample does not contain inhibitors • Purify the template again or dilute it • Make sure you are using the correct dye compatible with your qPCR platform (“ROX” or “no ROX”) • Increase the number of PCR cycles • Make sure the instrument is set up correctly for your experiment |
| Presence of fluorescence signal in negative control (without template) | • This is probably a contamination problem. Follow general instructions to avoid contamination. |
| Non-specific amplification | • Gradually increase the primer annealing temperature. • Pay attention to primer dimers. Adjust the primers if necessary |