Question: (Closed) Discrepancy between agarose gel and bioanalyzer after sonication for ChIP
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gravatar for augelloma
5.1 years ago by
augelloma0
augelloma0 wrote:

Hello, I was hoping someone could help me figure out the issue I am having with sonicated DNA for ChIP Seq.  I ran an initial sonication test in a bioruptor to optimize sheering size found that around 30 cycles looked to be in the right range (100-300bp) according to my agarose gel.  To get a more accurate measure of the DNA size and quantity I ran the same samples on a high sensitivity DNA bioanalyzer chip but got very different results.  While The DNA chip also picked up the smaller size fragments I saw on the agarose gel, it also showed a band smear near 1500-3000bp in nearly every sample (these bands were not present in the agarose gel).  I don't think it is a sensitivity issue because the band on the DNA ChiP is equal to or greater than the concentration of the lower band.  Has anyone ever seen this or know why I am getting to very different results?

 

Thanks

Mike

 

chip-seq • 2.0k views
ADD COMMENTlink modified 5.1 years ago • written 5.1 years ago by augelloma0

Hello augelloma!

We believe that this post does not fit the main topic of this site.

This is not a bioinformatics question, but a question that might be suitable for seqanswers.

For this reason we have closed your question. This allows us to keep the site focused on the topics that the community can help with.

If you disagree please tell us why in a reply below, we'll be happy to talk about it.

Cheers!

ADD REPLYlink written 5.1 years ago by Michael Dondrup48k

The X-scale is logarithmic, which makes the intensity of the high-molecular weight bands look much stronger than they appear on an agarose gel. This secondary peak is what we call 'overfixed' or 'undersonicated' chromatin, and re-sequencing experiments have shown that is doesn't really effect the results of the ChIP. The fixed chromatin is essentially from the same place as the unfixed chromatin (but different cells).

ADD REPLYlink written 5.1 years ago by John12k
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