The optimisation of a qPCR needs several steps:

  • Check for PCR inhibitors
  • Calculation of the primer efficiency
  • Estimation of the Linear Dynamic Range (LDR)
  • Optimisation of the primer concentration
  • Optimisation of the primer quantity
  • Optimisation of the consumables (plates, seals, pipet tips)
  • Optimisation of the size of the amplicon
  • Selection of the best-working qPCR master mix