Unconfigured Ad

Collapse
X
 
  • Time
  • Show
Clear All
new posts
  • jlove
    Member
    • Apr 2011
    • 49

    #1

    TruSeq libraries - double sizing?

    Has anyone else been getting PCR products that are exactly double the size of the template? I am seeing this occur with the TruSeq kits and also the KAPA enzyme. I have heard some theories - too much input (seems unlikely), samples clumping together, etc.

    I'd love to hear if other people have figured out what is causing this problem. It seems to be a new one over here.

    Thanks!
  • pbluescript
    Senior Member
    • Nov 2009
    • 224

    #2
    It is due to overcycling your PCRs, so it could also be due to too much input. I have attached an example of two libraries where I tried different numbers of PCR cycles. The gel shows 6-18 cycles in 2-cycle steps from left to right. In this case, I took the 8 cycle libraries.
    Here's another thread talking about this issue:
    Application of sequencing to RNA analysis (RNA-Seq, whole transcriptome, SAGE, expression analysis, novel organism mining, splice variants)
    Attached Files

    Comment

    • jlove
      Member
      • Apr 2011
      • 49

      #3
      Update: it was indeed over-cycling that created the problem. I believe that I was getting the amplicons partially hybridizing at the adapter regions and forming Ys which ran larger on the bioA. 10 cycles (vs 18) completely solved the problem. Thanks pbluescript for the ideas!

      Comment

      Latest Articles

      Collapse

      • SEQadmin2
        Multiomic Liquid Biopsy: What DNA/RNA Panels Reveal That DNA Alone Can’t
        by SEQadmin2



        Tumors contain complex mixtures of cell populations that evolve in response to treatment and host-specific factors. Tissue biopsies provide only static snapshots of a tumor’s state. But what if we could follow that evolution over time using a minimally invasive technique?

        Liquid biopsies can detect tumor-derived material circulating in the bloodstream, providing a way to study treatment response, detect emerging resistance, and discover new biomarkers. Circulating
        ...
        10-14-2026, 09:55 AM
      • SEQadmin2
        New Genomics Technologies Take Aim at Long-Standing Limits
        by SEQadmin2


        Researchers using sequencing and genomics tools often have to make trade-offs. They can choose between speed or scale, short reads or long-range information, or targeted panels or a view of the whole transcriptome. New technologies that have been released this year are built to address those tough choices.

        We asked six companies the same four questions to learn about their latest products. The new technologies bring a lot to the table, including rethinking sequencing
        ...
        09-28-2026, 10:25 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by SEQadmin2, Yesterday, 11:13 AM
      0 responses
      19 views
      0 reactions
      Last Post SEQadmin2  
      Started by SEQadmin2, Yesterday, 10:48 AM
      0 responses
      18 views
      0 reactions
      Last Post SEQadmin2  
      Started by SEQadmin2, 10-15-2026, 10:52 AM
      0 responses
      36 views
      0 reactions
      Last Post SEQadmin2  
      Started by SEQadmin2, 10-14-2026, 05:39 AM
      0 responses
      42 views
      0 reactions
      Last Post SEQadmin2  
      Working...