Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • For Nextera mate-pair kit, Is it possible to reduce the tagmentation reaction time?

    For Nextera mate-pair kit, Is it possible to reduce the tagmentation reaction time to get more long fragment, say 10kb? we have use this kit and found most of the smear is around2-7kb, but we want to do a large insert lib. does anyone try to reduce the tagmentation reaction time to get more long fragment, say 10kb? Thanks.

  • #2
    Have you tried using less enzyme? I think using less transposon would be a more effective way to increase fragment size than decreasing (the already short) reaction time.

    Comment


    • #3
      I'd say this LARGELY depends on the quality of your input DNA. Usually with very intact HMW DNA we've got a smear from 3-12kb and above. One of the samples, with slightly fragmented smeary DNA, was completely digested to below 1kb under the very same conditions as the samples mentioned previously.

      Comment


      • #4
        Reducing tagmentation enzyme

        We tried reduced amounts of tagmentation enzyme (4 ul or 8ul instead of 12 ul), expecting longer fragments, but it did not make any difference, unfortunately.

        This does not help the yield of relatively long tagmented DNA, but may help many users in terms of budget. But, probably we also need to know how to prepare self-made tagmentation buffer.
        Last edited by MesutOezil; 03-29-2014, 11:42 PM.

        Comment


        • #5
          Hi, I would like to ask if you sorted out your problem. I'm planing to use a Nextera Mate Pair Gel-Plus Sample Preparation kit for mate pair library. The problem is, that I don't have intact HMW DNA but only DNA from sorted and amplified wheat chromosome where the size is about 20 kbp. So I am interested if the decrease of time or amount of enzyme assure the less fragmentation but good tagging by adapters. Thanks. Katka

          Comment


          • #6
            Hi Katka,
            We tried only reduced tagmentation enzyme amounts (4 and 8 ul instead of 12ul), and it made some difference. We tested two species, and it seemed that different species show different effects upon varying the tagmentation condition. It is also of note that size distribution of tagmented DNA fragments should be measured in a reliable method.

            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, Yesterday, 06:37 PM
            0 responses
            11 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, Yesterday, 06:07 PM
            0 responses
            10 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 03-22-2024, 10:03 AM
            0 responses
            51 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