Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Removal of long fragmented DNA

    We are using the QSONICA Q800R machine for a while, in order to have fragmented gDNA at the size of 250-300bp. After sonication we tried Thermo Scientific PCR cleanup kit, and then applied the NEBNext DNA library prep kit for Illumina. After size selection of adaptor – ligated DNA, there are still unwanted large fragments.

    Do you have any ideas to remove this unwanted lenghts?

    Thanks in advance,
    Adi

  • #2
    Hi Adiza, please search for "Ampure XP bead upper cut" protocols.
    An alternative is gel based size selection; manually or with a Pippen Prep system.

    Comment


    • #3
      Hi luc

      are you sure that the "upper cut" protocol fits to gDNA sonicated samples before turning it into libraries?
      I've tried the E-Gel method (if that was your intention) though it's too wasteful in the step of sonicated samples...

      Comment


      • #4
        250-300 bp is a very narrow cut and requires Pippin or similar automatic size selection instrument. Size selection with bead would result in broader size range.

        Large fragments do not affect sequencing output and results unless for a specific purpose you want tighter size range.

        If you have done size selection after adapter ligation and run it on TapeStation or Bioanalyzer you will get wrong size and possibly double peak. You should check the size after PCR purification.

        Comment


        • #5
          Originally posted by luc View Post
          Hi Adiza, please search for "Ampure XP bead upper cut" protocols.
          This is what we use as well. It's pretty good at removing higher length fragments.

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Strategies for Sequencing Challenging Samples
            by seqadmin


            Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
            03-22-2024, 06:39 AM
          • seqadmin
            Techniques and Challenges in Conservation Genomics
            by seqadmin



            The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

            Avian Conservation
            Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
            03-08-2024, 10:41 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, 03-27-2024, 06:37 PM
          0 responses
          12 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-27-2024, 06:07 PM
          0 responses
          11 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          53 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          68 views
          0 likes
          Last Post seqadmin  
          Working...
          X