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Old 06-13-2016, 01:16 PM   #1
zgene
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Location: Asia

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Default Bioanalyzer for ChIPed DNA

Dear All,
I have a question regarding bioanalyzer for ChIPed DNA.
When I do bioanalyzer for the input DNA, I get peaks at the proper range in bioanalyzer.
But, for the immunoprecipitated samples, I got no peaks at all or strange peaks. I even do not have the marker peaks!
I should mention that the concentration of ChIPed DNA was as low as 0.3 ng/Ál (measured by Qubit HS kit) and I used 1 Ál for bioanalyzer. So, I expected very low signals in bioanalyzer, but literally got nothing.
These samples were loaded in a same bioanalyzer chip used for some other library prepped samples and they all worked well, which shows that there was no problem in bioanalyzer chip or experiment.
Can anyone help me how to explain this?
I am totally confused and cannot figure out what I should do!
In the attached you can find a gel photo of the sonicated chromatin as well as two bioanalyzer graphs of two different ChIPed samples.

waiting for your replies...
Ladder.png

Attachment 4371

ChIPed Sample12.png

gel photo after sonication.jpg
Attached Images
File Type: png ChIPed Sample1.png (21.5 KB, 9 views)

Last edited by zgene; 06-13-2016 at 01:21 PM.
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Old 06-14-2016, 04:27 AM   #2
nucacidhunter
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It seems that one of the wells was not loaded with marker and the strange peak in other well could be due to contaminants from immunoprecipitation step. I could not figure which Bioanalyser trace is from input DNA.
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Old 06-14-2016, 01:41 PM   #3
zgene
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Quote:
Originally Posted by nucacidhunter View Post
It seems that one of the wells was not loaded with marker and the strange peak in other well could be due to contaminants from immunoprecipitation step. I could not figure which Bioanalyser trace is from input DNA.

Dear nucacidhunter,

Thanks for the reply.
Marker was loaded in all the wells.
I did not attach the Input traces there, there was only a gel photo for that.
The Input traces are now attached here.
input.png
For the contaimnation, I should say that I used the columns available in the kit (Abcam high sensitivity ChIP kit) which includes 3 washing steps with ethanol after DNA binding to the column.
Do you think that its better to replace this step with qiagen kit to get less contaminants?
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Old 06-14-2016, 03:00 PM   #4
nucacidhunter
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I would contact kit supplier before trying another column for clean up. It could be result of some inefficiency at steps prior to clean up. Sample showing no trace in BA should be rerun to make sure that it is reproducible.
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