Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • PBMC samples chromatin extraction - low concentration

    Hello,

    I have been trying to extract chromatin from an immune system population, but the DNA concentration levels that I've been getting are extremely underwhelming.

    Namely, from 1 million to 3 million cells I am getting concentrations of around 0.525ng/uL to 2.08ng/uL of chromatin in 50uL of SDS lysis buffer (1%SDS, 100mM EDTA, 50mM Tris pH 8.0), measured on Qubit with the dsDNA HS kit.
    Fixation was done with Formaldehyde 37% for 10 minutes.

    Anyone with experience on extracting chromatin from immune cells? I need to know if these are normal concentration values for these cells, because I am at a loss already.

  • #2
    Update: Tested a new formaldehyde to compare with the old one, but still got around the same concentrations. Tested this in total thymocytes instead of doing it on sorted cells. Still no improvement.

    Update 2: Sonicated these samples. After sonication the concentration increased to the expected values. Uh. Apparently the sonication is required not only to fragment DDNA but to actually release from the cell debris?
    Last edited by Rosmano; 01-13-2022, 10:40 AM.

    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
    12 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