Troubleshooting a real-time PCR assay is an essential part of assay optimization and validation. Researchers can significantly reduce erroneous data and downtime by going through this checklist:

  • Ensure that primer-dimers are not contributing to signal or poor efficiency
  • Take steps to minimize PCR inhibition and poor reaction efficiency, for example by re-purifying the template or using an appropriate magnesium concentration
  • Confirm appropriate software and instrument analysis settings
  • Make standard curve validation the final step in the reaction assessment process
  • Understand how to address efficiencies below 90% versus those above 110%
  • Mitigate the sources of no amplification
  • Identify factors that most seriously affect data accuracy, particularly amplification curve baseline linearity, baseline range settings, threshold, and reference dyes

The culprits of unsuccessful real-time PCR assays are many and varied. Identifying the root causes of analysis issues is the first step in successful troubleshooting.

In addition to troubleshooting tips, our “Real-time PCR handbook” is the definitive source for all things related to real-time PCR, including:

  • Basics of real-time PCR
  • Experimental design
  • Plate preparation
  • Data analysis
  • Digital PCR

Download your FREE copy of the “Real-time PCR handbook” today!

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