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  • Nextera DNA sample preperation problem

    Hello everyone my first time on here
    Can any one help
    Im using the Nextera sample preperation kit.
    My problem in some samples
    the DNA does not sheer properly leaving me with very large Fragments well over 1kb

    The DNA is WGA samples from single cells

    Has anyone encountered this problem and if so did yoou find a solution


    Hope you can help Derek

  • #2
    Hey Derek,

    How do you quantify your amount of input DNA, maybe you are putting in too much DNA.
    You can also try to use less input DNA. Try to set up a reaction with 25ng of input DNA.

    Hope that helps.

    Comment


    • #3
      May be caused by the volume you applied, like using Covaris, some unsuitable volumes in the Covaris shearing step can cause the air gap contributing to "phase separation" that results in the presence of larger fragments in the sheared DNA.
      Last edited by weigrc; 06-26-2012, 06:17 PM.

      Comment


      • #4
        Originally posted by weigrc View Post
        May be caused by the volume you applied, like using Covaris, some unsuitable volumes in the Covaris shearing step can cause the air gap contributing to "phase separation" that results in the presence of larger fragments in the sheared DNA.
        Nextera uses a "tamed" transposase to break strands and add end-adapters. So air gaps will not be an issue.

        The WGA is probably the issue. Most of them create complex branched double-stranded structures of extremely high molecular weight. My wanton speculation would be that the transposase likes the WGS junction points where there is a single-stranded section? Then those areas would get hammered while the double stranded segments would get few insertions.

        Seems like you could overcome this bias (or most other sorts of bias) by increasing the incubation time.

        --
        Phillip

        Comment


        • #5
          Derek

          Hi everyone thanks for the replies
          I know my DNA conc was correct
          We thought here in Liverpool it was a WGA problem as pmiguel points out
          Epi center suggest its a phenomenom called bird nesting were the samples are sticking together and look like longer fragments on bioanalyser traces when in fact they are the correct size.

          They suggest that the denaturning step during bridge PCR will eliminate the tangle and the average size will settle back to 200 400bp

          if you interseted you can find it here

          Blogger is a blog publishing tool from Google for easily sharing your thoughts with the world. Blogger makes it simple to post text, photos and video onto your personal or team blog.


          thanks for your help guys

          ta Derek

          Comment

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