Biology Forum Molecular Biology Nested PCR

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    • #9818
      ishla
      Participant

      Can you use one of the primers you have used in the first round of your nested PCR in the second round along with a new primer situated within the first round product???

    • #84944
      Cat
      Participant

      Yes you can, but expect artifacts along with your expected product.

    • #84964
      dipjyoti
      Participant

      Nested PCR means that two pairs of PCR primers were used for a single locus.
      The selection of alternative and similar primer binding sites gives a selection of products, only one containing the intended sequence. The product from the first reaction undergoes a second run with the second set of primers.
      It is very unlikely that any of the unwanted PCR products contain binding sites for both the new primers, ensuring the product from the second PCR has little contamination from unwanted products of primer mis-binding and alternative primer target sequences.

    • #84988
      Cat
      Participant

      Ishla is actually doing semi-nested PCR: one primer is the same in both rounds. That means that single primer amplification artifacts are possible.

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