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  • Bead removal of <1000 bp from LARGE fragments

    Hi,

    Trying to prep some very large fragment libraries (>50 kbp), and I've isolated the gDNA using a BluePippin, but when I use beads (Promega ProNex) to clean up, or rather, remove the small fragments (<1000 bp), I get low yield of the input 50 kbp gDNA.

    Are there specific beads that are better for this? AMPure XP PB? Suggestions?

    Thanks,
    Andor

  • #2
    The beads are likely all very similar. You could try to elute for a much longer time than usual for short fragments.

    Comment


    • #3
      No, I don't want the small fragments. I want only fragments LARGER than 1000 bp.

      In essence, I want to eliminate any small (unseen) fragments that may unduly influence a nanopore/ONT minION library prep.

      Thanks

      Comment


      • #4
        Loss of large fragments mostly can be due to inefficient binding. longer incubation of bead-DNA solution on a rotating wheel should increase recovery.
        Quantifying supernatant with PicoGreen or Qubit dsDNA HS assay can point if this is the case.

        Circulomics short read eliminator kit is also a good choice.

        Comment


        • #5
          You misunderstood me: Long fragments will require a longer time to elute/dissolve from the beads compared to shorter fragments.

          Originally posted by cement_head View Post
          No, I don't want the small fragments. I want only fragments LARGER than 1000 bp.

          In essence, I want to eliminate any small (unseen) fragments that may unduly influence a nanopore/ONT minION library prep.

          Thanks

          Comment

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