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  • PCR conditions for PE adapters

    Hi,

    I was wondering if anyone could help with the optimal cycling conditions for the post-adapter-ligated amplification step (using PE adapters) in the Sure Select Target Enrichment system? Given the short and long PCR primers required, is a sort of 2-step PCR required?

    Additionally if one were to use the "homemade" adapters, are the adapters required to be pre-annealed before the amplification? How are these "Y" -shaped adapters generated?

    Any help would be great,

    Thanks,
    Luke.

  • #2
    Hi Luke, I'm also looking for PCR conditions for the post-adapter ligation step. I ordered single stranded HPLC purified adapters and annealed them before ligating to the sequence. I'm not sure how it has worked yet as I haven't done the PCR.

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    • #3
      I recently tried annealing the adapters together, amplifying, and running the products on a agarose gel.. i obtained 2 amplification products... one approximately 150bp and one slightly smaller.. i've gel extracted these bands but whether they do represent genomic-adapter fragments remains to be seen.. I'll keep at it, and post any progress.

      Cheers,
      Luke

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      • #4
        y-shaped adapters

        Hi Luke,

        have you made any progress with creating the y-shaped adapters?
        We sequenced them ourselves as well and I wonder if a preannaeling
        process is necessary because Illumina supplies them in a mix already.
        So I have the same issue than you have.
        Did you recieve any information yet?
        Thanks!!!!!
        Theresa

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