QPCR Troubleshooting Guide

QPCR Troubleshooting Guide

Problem Possible solution
Low or no fluorescence or high CtIf 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