Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • No DNA in ChIP IgG Control - Strange?

    Hi Guys,

    Thanks in advance for your help!

    Background:
    It's my first time doing ChIP. I am testing a native ChIP protocol with MNase digestion using Protein A/G beads and H3K4Me3 and H3K27Me3. Because my samples are mononucleosomes (140bp) I didn't do qPCR prior to library preparation because of the difficulty of designing primers for such small fragments. I used 30,000 Cells (10,000 per IP).

    Problem:
    Although there is product in my H3K4Me3 and H3K27Me3 IP's, I have no product in my IgG sample (as detectable by HS Bioanalyzer). In my naive knowledge of ChIp I am tempted to say this is a great result as my IgG is a negative control and should hypothetically have pulled down no sample but someone else in my lab who has done X-linked ChIP using Diagenode's kit has a fair deal of product in her H3K's AND IgG's.

    Any ideas as to the difference? I used Rabbit IgG instead of mouse (my cells are murine) - would that make a difference?

    I've attached my Bioanalyzer traces.
    Side note: My Input control is quite low - the main kit I am modifying for this N-ChIP suggests using 10ul from a 100ul dilution of chromatin for Input control. Instead, I pooled 4ul of each Input dilution prior to immunoprecipitation to result in 12ul of Input control for library prep. Do you think this is a good method for Input control? I am quite worried that the concentration is lower than my IPs - although it makes sense why it is (due to the lower input) I am not sure this is a good experimental design or if it is normal for all ChIPs? Would a better design be splitting my chromatin in 4 - 3 x 100ul for IPs and 1x 100ul for Input control? That way the input is the same - or does that not matter since I only pull down a percentage of DNA in each 100ul IP vs ''100%'' of on input control?

    Sorry for the rambling questions!
    Attached Files

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