Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • NEBNext Adapter Ligation Efficiency

    Hello.

    Has anyone tried "NEBNext Multiplex Oligos for Illumina" to generate sequencing libraries? I have been experiencing a very poor ligation efficiency of NEBNext (hairpin) adapter. I ligated end-repaired/3'A added 1 ug of sonicated gDNA to NEBNext adapters or TruSeq adapters and amplified them by 18 cycles of PCR. The result was frustrating. The NEB one was barely amplified, whether "USER" was added or not, compared to TruSeq samples. Is there something I should've consider for the hairpin adapter ligation? If any of you has experienced this, help me please!!!


    <NEBNext Oligo kit I used>


  • #2
    When I use these kits, which I really like, I just use the TruSeq adapters. Unless you specifically NEED the NEBNext adapters, why not just use the TruSeq?

    Comment


    • #3
      Hi,

      we always use the NEB adaptor and in our hands it is clearly superior in terms of ligation efficiency compared to the Standard Y-shaped adaptors.

      I don't know the current version of the protocol. Back in the days, the USER digest was done in the first step of the PCR.
      We changed that in a way that we do the USER digest for 30' to 1h at 37°C, purify the DNA with Ampure beads and then perform the PCR.

      That clearly improved the yield of our libraries.

      Comment


      • #4
        In my previous lab, we found that with certain plastics, the adaptors were binding to it. When doing the dilution, we were using nuclease-free water sometimes and not the suggested 10 mM Tris-HCl and that was also affecting how much the adaptors bound to the plastic. We found this out by just putting the adaptors into a plate (BioRad hardshell) and waiting for various lengths of time before running on a BioA chip. The longer it sat, the less showed up.

        Comment


        • #5
          If you don't UDG+Exo it well, it won't PCR at all. nanos has it right, ensure the ideal conditions for USER.

          Comment


          • #6
            If you are starting with one microgram of DNA you shouldn't need PCR at all, much less 18 cycles. Also, at that amount of starting material differences in ligation efficiency probably don't matter much, so I also recommend going with TruSeq-style adapters so that you don't need a USER step. And you don't necessarily need to get these from Illumina, Bioo Scientific offers many Y-shaped adapter options, including PCR free, which I think would be best if you are starting with one microgram of DNA. As a disclaimer, I work for Bioo Scientific.

            Comment


            • #7
              If you want to go for Y-shaped adaptors, you can also just order the two strands as oligo (best is HPLC purified) and perform a simple annealing reaction before you do the ligation. We did that for a long time and it worked quite well (maybe a little less efficient than the NEB adaptors)

              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
              10 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 06:07 PM
              0 responses
              9 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-22-2024, 10:03 AM
              0 responses
              50 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-21-2024, 07:32 AM
              0 responses
              67 views
              0 likes
              Last Post seqadmin  
              Working...
              X