Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Disagreement between bioanalyzer and agarose gel for PCR amplicon library

    Hello,

    We are setting up for an illumina MiSeq run using a PCR amplicon-based library (adding the Illumina adaptors using nested PCR). Our results look quite clean via agarose gel (expected size ~310nt), but when our facility ran bioanalyzer we had quite variable results (ranging between 400 and 500nt, depending on the sample). There are seven samples, each with a different barcode, labeled 1-7.

    I'm attaching both the gel and the bioanalyzer report - has anyone seen anything like this before?

    Thanks,
    Michael
    Attached Files

  • #2
    I can only speculate. Maybe the agarose gel was run either without EtBr, then post run stained, or no EtBr was put in the gel or loading solution, but was put in the buffer? In either of these cases, the products would migrate mostly or entirely in the absence of EtBr.

    Why is that important? Well, I have not tested this myself, but Dave Cook at Sage Science told me that in the absence of EtBr (or other DNA binding dyes) the difference in mobility between fully double-stranded amplicons, and "bubble products" (double stranded only at the adapter ends) does not happen. I.e. everything basically runs at the "correct" size.

    So, if this is the issue you see, your amplicons are fine, but consist of variable amounts of normal and bubble-products. The Bioanalyzer chip faithfully displays the difference with the bubble products migrating slower than their true molecular weight.

    Again, just a guess.

    --
    Phillip

    Comment


    • #3
      Make sure you aren't overloading the chip. The first 3-5 samples look really hot relative to the standard. You could also try a DNA1000 chip since your products are so small.

      Comment


      • #4
        Hi Michael--
        I was curious how this worked out for you. I recently prepared an amplicon library for TnSeq and had a similar discrepancy between my gel (the expected 190-bp band) and my bioanalyzer results (around 220 bp). I couldn't figure out why this happened.
        -Tom

        Comment


        • #5
          I think this has to do with the mobility of the y-forked adapters. Agarose is more difficult for migration because it's a solid phase, whereas the Bioanalyzer chip's gel matrix is much more fluid. I think the DNA 7500 chip gives the best representation of the "real" size.

          Comment

          Latest Articles

          Collapse

          • seqadmin
            Genetic Variation in Immunogenetics and Antibody Diversity
            by seqadmin



            The field of immunogenetics explores how genetic variations influence immune responses and susceptibility to disease. In a recent SEQanswers webinar, Oscar Rodriguez, Ph.D., Postdoctoral Researcher at the University of Louisville, and Ruben Martínez Barricarte, Ph.D., Assistant Professor of Medicine at Vanderbilt University, shared recent advancements in immunogenetics. This article discusses their research on genetic variation in antibody loci, antibody production processes,...
            Today, 07:24 PM
          • seqadmin
            Choosing Between NGS and qPCR
            by seqadmin



            Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...
            10-18-2024, 07:11 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, 11-01-2024, 06:09 AM
          0 responses
          24 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 10-30-2024, 05:31 AM
          0 responses
          21 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 10-24-2024, 06:58 AM
          0 responses
          25 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 10-23-2024, 08:43 AM
          0 responses
          56 views
          0 likes
          Last Post seqadmin  
          Working...
          X