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Why do my primers have deleted 3' bases in the amplified fragment when I use a proofreading enzyme for PCR?

Proofreading polymerases (Pfu, Diamond Taq®, etc) have been shown to remove 3' bases from PCR primers (see Nucl Acids Res 20(14):3551-3554, for more information). If you experience this problem, you can protect your primer from degradation by adding one or more phosphorothioate bonds to the 3' end of your primer. Also make sure to add the enzyme last, after the addition of dNTPs, in your PCR to minimize the risk of this issue taking place.
  • Pfu
  • polymerse
  • proofreading
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