Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • ChIP seq sample prep for Illumina

    Hi Everyone, I'm hoping someone can give some advice on ChIP enriched DNA. Does anyone try and quantify this by nanodrop/picogreen before starting the Illumina sample prep protocol (the protocol recommends starting with approximately 10ng DNA)? We have had difficulty in quantifying samples reliably. Is anyone doing ChIP seq regularly that can help?
    Thanks.

    EDIT : Sorry, now posted in Illumina section!
    Last edited by athos; 09-19-2008, 01:11 AM.

  • #2
    It's possible but not practical to quantify your IP DNA. To do it you need to either resuspend it in a small volume (10-15uL) and use a nanodrop or use a SYBR assay. In practice we only do this occasionally, such as when we are testing a new antibody or if we suspect a problem. Beyond that we just use the yield after LM-PCR as our first QC checkpoint.

    I wouldnt' worry about getting exactly 10ng input. What is more important is to empirically optimize the concentration of adaptor that works in your hands. The illumina protocol calls for something like a 1:10 ratio of insert to adaptor but there is a wide range of IP yields dependent on protocol, antibody and phase of the moon so you really need to do the titration yourself. We find for ChIP-seq that something between a 10 and 100 fold dilution of the adators in the genomic DNA kit works for us.

    Comment


    • #3
      Because of the lower concentration of the ChIP DNA samples, it is not accurate to check it by nanodrop, it's better to use picoGreen stain.
      Although the Illumina's protocol recommend 10ng is enough, many companies could not realize this for library preparation. And you also could not get a good result to use only 10ng. It's better to use more than 100ng. Gook luck!

      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
      10 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      9 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
      67 views
      0 likes
      Last Post seqadmin  
      Working...
      X