Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Strange higher band in Bioanalyzer after size selection

    Hi all,
    I just got the data back from 7 samples Ive run on the Bioanalyser (see attached file). All samples have been prepared with the illumina Paired-End kit and I size selected the libraries around 350pb (+/- 25bp). In more or less all the samples I see an additional band around 500-550pb.

    Basically, I have no idea what this band is since the only step after the size selection is gel extraction with the qiagen kit.

    Have any of you ever seen something similar? I was wondering whether it could be somekind of dimers that have been forming?

    Any ideas about what it could be, how to test it and how to get rid of it will be greatly appreciated.

    Thanks,

    Jesper
    Attached Files

  • #2
    Hi,
    I think there may be some problems when you doing your Gel cutting. Maybe your gel cutting is not very sharp and straight.

    From my view, I do not think it is the dimer. The peak around 150kb should be the primer or dimer or something.

    I usually do Gel extraction again after PCR reaction.

    Hope these can work for you.

    Comment


    • #3
      There was some discussion about this on another thread. The thought was that it may be due to overamplification of the adaptor ligated fragments. I use the Invitrogen E-Gel size select system to isolate libraries both before and after PCR amplfication. I also saw a very light higher molecular weight band on the gel ( as well as lower molecular weight primer dimers and adaptor complexes) but after analysis on a bioanalzer, all that was there was a tight band around 250 bp that represented my library. I'm waiting to get back my sequence.

      Comment


      • #4
        This is not due to the size selection and it is not primer-dimer. We see this as well and think it has something to do with how the DNA is migrating. One thing we have noticed is that you need to take in to account the additional peak. Else, your cluster numbers will be too high.

        Comment


        • #5
          this protocol is the epitome of unstandardized trash.

          Comment


          • #6
            Just to throw it out there for comment, but the higher peak may represent single stranded PCR product, which does not run correctly on the bioanalyzer. It occurs when there is too much DNA added to the final PCR reaction, and then there is not enough primers in the solution for the last 72C 5 minute extension. We saw it to a small extent with Agilent sureselect, but now see it a lot more with Nimblegen, probably because the Nimblegen hybridization is more efficient at pulling down DNA fragments.

            Comment


            • #7
              Originally posted by JHU-ChIPmaniac View Post
              There was some discussion about this on another thread. The thought was that it may be due to overamplification of the adaptor ligated fragments. I use the Invitrogen E-Gel size select system to isolate libraries both before and after PCR amplfication. I also saw a very light higher molecular weight band on the gel ( as well as lower molecular weight primer dimers and adaptor complexes) but after analysis on a bioanalzer, all that was there was a tight band around 250 bp that represented my library. I'm waiting to get back my sequence.
              What is your results? I also see a 250 bp band in my library.

              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
              8 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, Yesterday, 06:07 PM
              0 responses
              8 views
              0 likes
              Last Post seqadmin  
              Started by seqadmin, 03-22-2024, 10:03 AM
              0 responses
              49 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