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Why does my extracted DNA fail to amplify properly? I notice a lot of PCR inhibition and/or Ct values that make no sense.

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PCR inhibition is often observed when high amounts of FFPE-extracted template DNA are used. The inhibition is not usually due to the presence of contaminants but results from residual chemical modifications and damage in the DNA itself. Several simple adjustments to the PCR protocol can overcome this issue. First, the amount of template DNA should be decreased. Second, the amount of PCR polymerase should be increased by 2-4X. Third, the annealing and extension times should be extended. Fourth, the amount of dNTPs can be increased.

An in-depth discussion of this issue is found in Dietrich et al. (2013), PLoS ONE 8(10): e77771.

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