Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Accidentally eluted with x100 Tris

    Hi All,

    I have just realised that at the end of the Ampure clean-up, I eluted with x100 Tris buffer by accident. Is this the source of my problems? And is there any way to solve this??
    I have 120 human feacal samples which I am preparing for MiSeq at the moment. I am following the Illumina protocol and have the first PCR and clean-up completed (with very good PCR products).

    I attempted to run the first plate with the Nextera library prep primers yesterday and when I ran the gel all I got back was a very faint band around where the first (16s amplicon) PCR product was (550bp) and no bands at all where they should be (630bp).

    After rerunning 4 of my samples on a different PCR block and getting the same result, I gave a couple samples to my colleague who was doing a Nextera library prep and offered to try them. She got the same result and her prep didnt work.

    Thank you!

  • #2
    Are you running the Illumina metagenomics protocol, and do you mean you eluted your first PCR rxn in 100x Tris? That's a lot of Tris to be putting into your second PCR rxn!

    If this is the case, I'd try "recleaning" a couple samples from your first reaction and eluting into the proper Tris concentration. Then try the second PCR again.

    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
    66 views
    0 likes
    Last Post seqadmin  
    Working...
    X