Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Yeast ChIP-seq; Sonication/Bioanalyzer question

    Hi,
    I've just started doing ChIP-seq, and am having difficulty getting my INPUT and IP DNA fragment sizes in the proper size range for Illumina sequencing. My ChIP protocol seems to work fine, as I can show enrichment of a 360bp region surrounding a known binding site in my IP sample compared to the INPUT sample by pcr.

    However, our Bioanalyzer reports that the size fragmentation is too small for the library prep. This seems to indicate that I'm over-sonicating the samples. I've been following a protocol published earlier this year by Mike Snyder's lab http://dx.doi.org/10.1016/S0076-6879(10)70004-5. In this protocol the sonication procedure uses a Branson Digital Sonifier @ 5x30sec @ power 50.

    In our lab, we have a Branson Analog Sonifier and I've tried sonicating 6x10sec @ power 20 and 3x10sec @ power 20. In both cases, the bioanalyzer indicates that my DNA size fragmentation is mostly <100bp. However, when I run the INPUT DNA on a 2% Agarose gel, I see a smear with the greatest DNA concentration ~1,000bp and no DNA below 100bp.
    Last edited by AaronS; 01-14-2011, 11:13 AM.

  • #2
    Hi AaronS.
    I think that the not real result is the agarose gel. The bioanalyzer is more specific than gel, where you have less resolution to see small DNA quantity and migration is not the same (the upper marker of a DNA HS is 10380bp, but you see all the largest fragmented sizes grouped around 1500bp). If you try with DNA 1000 or 7500, you will have the same result (you will see all large sizes grouped around 1500bp too).
    I think that the smear of fragmented sizes that you see in bioanalyzer, you can't see in agarose...you only can see sizes with more concentration (usually largest sizes but they are in correct size compared with marker, not as occurs in bioanalyzer).
    The sample that you see in bioanalyzer at 100bp, may be DNA degradation of Chip and Input. If you purify the samples by column (Qiaquick for example), and run again the samples at bioanalyzer, you will not see anything less than 150bp.
    You can try to fragment more Inputs with different times. Then you can run it in a bioanalyzer and know if you are fragment too much your samples.
    It can be a protein contamination from previous step too (it could explain this results).
    In order to can see better the DNA purified by column, you can put 2-3ul evaporated (quantitative range of DNA HS is up 0.5ng/ul).
    I hope you can solve this problem with my comments.
    Regards.

    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
    11 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, Yesterday, 06:07 PM
    0 responses
    10 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-22-2024, 10:03 AM
    0 responses
    51 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 03-21-2024, 07:32 AM
    0 responses
    68 views
    0 likes
    Last Post seqadmin  
    Working...
    X