Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • ChIP-Seq mouse embryonic sample prep

    I am planning for to do a ChIP-seq with chromatin prepared from embryonic sample(E7.5 mouse embryo). For fixing the protein-DNA complex I have used 1% Formaldehyde @ 37 C for 10min.
    Sonication cycle= 30 seconds ON and 30 seconds
    No of cycles= 60
    But still after such high number of cycles I am unable to get the desired fragment size of 200-400bp for ChIP seq.
    What could be the possible modification I could do to improve the current situation?

  • #2
    ES cells are much more difficult to crosslink and sonicate properly. Their chromatin is much more compact. You first need to optimize this. Start by varying the time and temperature of the crosslinking. 10 min at 37C is obviously way too much.

    If formaldehyde ends up being too strong of a crosslinker for your ChIP target you may need to first use EGS or DSG then finish with a low concentration of FA. There are a few protocols that have been published using these alterantive crosslinkers.

    Comment


    • #3
      If you can not get this to work Active Motif has a ChIP-Seq Service that does the whole project. We have done lots of ES ChIP-Seq.

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Current Approaches to Protein Sequencing
        by seqadmin


        Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
        04-04-2024, 04:25 PM
      • 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

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 04-11-2024, 12:08 PM
      0 responses
      18 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 10:19 PM
      0 responses
      22 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-10-2024, 09:21 AM
      0 responses
      17 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 04-04-2024, 09:00 AM
      0 responses
      49 views
      0 likes
      Last Post seqadmin  
      Working...
      X