Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Nextera Success?

    Hello,

    I'm trying to use an old Nextera kit from before the company was acquired by Illumina. I have tried using the kit's HMW buffer on 15 ng of the included lambda control DNA.

    After tagmentation and limited-cycle PCR, I get the attached size distribution. This is not anything like the size distribution I expected from the rather terse instruction manual, nor is it similar to the ones pictured 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.


    Would the correct interpretation be that tagmentation failed or worked only poorly, or am I looking at something else entirely here? Besides bioanalyzer traces, what are other standard controls I might do to validate my library? I've thought about trying to PCR up random fragments, but it seems difficult. I'll appreciate any input at all.

    Carl Wivagg
    Attached Files

  • #2
    The kit has got to be expired by now if it was made before Illumina acquired Epicentre a couple years ago.

    Comment


    • #3
      Thank you for this observation... it may be expired. I am more interested, though, in interpreting this result to determine what component of the kit has failed. I was hoping someone who has a bit of expertise in library preparation might be willing to offer a guess, or suggest controls. I would rather learn to troubleshoot on an expired kit than waste expensive new reagents.

      Carl

      Comment


      • #4
        Hi, do you have a lot number for that kit? Expiration is certainly the main consideration and the Transposomes (Nextera Enzyme Mix) probably went bad.

        Comment


        • #5
          I agree...it has to be the enzyme...everything else is buffer.

          Comment


          • #6
            If you really want to use this kit you could try using only 1-2ng input to see if the fragment size distribution gets in the 200-300 range.

            Comment


            • #7
              If you buy a new kit, and run conditions where you replace one of the new reagents with one of the old reagents...then you can see which one is the trouble maker. It sounds like you need a new kit anyways.

              what do you mean, "standard controls to validate your library"?

              Comment


              • #8
                I believe that Nextera has short shelf life due to short half-life of transposon complex. If the kit is not fresh, you need to optimize fragmentation conditions again, such as using more enzyme etc. I went back to regular library prep protocol. Transposase(s) does not seem to be very stable.

                Comment


                • #9
                  Originally posted by SeqR&D View Post
                  If you buy a new kit, and run conditions where you replace one of the new reagents with one of the old reagents...then you can see which one is the trouble maker. It sounds like you need a new kit anyways.

                  what do you mean, "standard controls to validate your library"?
                  Regarding controls, I am wondering if there is anything I can do beyond a Bioanalyzer run. Do people try to TOPO-clone fragments to verify proper end-tagging, or anything like that?

                  Thank you everyone for your thoughts. I have since bought another Nextera kit and observed much better fragmentation. I am thinking about going back to the original kit and trying a much longer incubation to see if I get any fragmentation, as one responder suggested, since the consensus seems to be that the transposome is probably decayed.

                  Comment

                  Latest Articles

                  Collapse

                  • seqadmin
                    Current Approaches to Protein Sequencing
                    by seqadmin


                    Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
                    04-04-2024, 04:25 PM
                  • 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

                  ad_right_rmr

                  Collapse

                  News

                  Collapse

                  Topics Statistics Last Post
                  Started by seqadmin, 04-11-2024, 12:08 PM
                  0 responses
                  11 views
                  0 likes
                  Last Post seqadmin  
                  Started by seqadmin, 04-10-2024, 10:19 PM
                  0 responses
                  17 views
                  0 likes
                  Last Post seqadmin  
                  Started by seqadmin, 04-10-2024, 09:21 AM
                  0 responses
                  14 views
                  0 likes
                  Last Post seqadmin  
                  Started by seqadmin, 04-04-2024, 09:00 AM
                  0 responses
                  43 views
                  0 likes
                  Last Post seqadmin  
                  Working...
                  X