Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Beads sticking to tube during Ribo-Zero protocol

    Hi all. I noticed that the Ribo-Zero magnetic beads tend to stick to the sides of the tube after you add your RNA sample and mix with pipetting and vortexing, and that this effect carries on through the end. When I finally pipette the 90 uL of sample from the magnetic stand, I usually just am very careful to guide the tip to the liquid without touching any sides since there will be beads stuck to it. However, recently I did the protocol and the sides of the tubes looked much cleaner than normal. My only thoughts are that I used a different brand of PCR tube during the incubation with the removal solution... any other ideas for why this may have changed? Or what your experiences are using Ribo-Zero?
    - Gina

  • #2
    We recently used ribo-zero for metabacteria and have >50% of our reads mapping to bacillus subtilis rRNA and e. coli rRNA. It appears that the capture failed for some reason. Does anyone perform QC to check for probe leakage? Anyone else experienced probe leakage? Its pretty frustrating.

    Comment


    • #3
      We carried the libraries through despite the odd appearance of the beads which seems to be not unusual after all, and got less than 99.5% rRNA contamination with Oryza sativa using the plant leaf kit. I can't speak for the other kits and your application though. It would be worth it to contact Epicentre. They are very good at helping you troubleshoot until the protocol is working for you (providing some reagents for your trouble in the process).
      - Gina

      Comment


      • #4
        Thanks for the reply. We've been using the metabacteria kit for awhile with good results so our recent experience is an outlier however our level of confidence in the product has decreased. Beyond basics such as making sure the beads are used and stored under the recommended conditions, Epicentre tech support was unable to help us. We have the contaminating sequences so we'll probably use PCR to QC the ribo-zero treated RNA for the capture oligos.

        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, 03-27-2024, 06:37 PM
        0 responses
        13 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-27-2024, 06:07 PM
        0 responses
        11 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-22-2024, 10:03 AM
        0 responses
        53 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-21-2024, 07:32 AM
        0 responses
        69 views
        0 likes
        Last Post seqadmin  
        Working...
        X