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  • Gel-size selection in NimbleGen exon capture

    Hey,

    just wondering if anyone has tried omiting the gel-size selection following Illumina adaptor ligation prior to commemcing the LM-PCR step in the NimbleGen in-solution Exon capture protocol?

    If so does the residual adaptor dimers have any marked effect on the downstream data?

    Cheers,

    Mitch.

  • #2
    Hi,mitchels
    I am tring the gel-size selection following Illumina adaptor ligation prior to commemcing the LM-PCR step in the NimbleGen in-solution Exon capture protocol。But we can't get enough PCR products after LM-PCR(8 cycles)。Are you successfully with the protocal?
    Thank you!

    Comment


    • #3
      I have found that the skipping the gel size selection can result in increased primer dimers in the pre-hybridization PCR. The Illumina protocols suggest an extra magnetic bead clean after ligation, this seems to help with the PCR.

      Comment


      • #4
        Size selection is definitely not needed by gel, but a bead-based size selection to get rid of adaptor dimer is really helpful. A single 1x bead purification will get rid of stuff below 200nt prior to library amplification.
        HudsonAlpha Institute for Biotechnology
        http://www.hudsonalpha.org/gsl

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