It seems you have over-sonicated your samples. I would say, the fragment seems to be around50bp rather than 200bp. First, you need to run your samples again on 1% gel with a ladder contain 50bp, so you will see your exact fragment size. Furthermore, in such senario, you need to start taking up your sample after 1 cycle or 2 cycle, and dont wait for 5 cycles. You need to optimize your sonication by limiting the cycle numbers, as other parameters look ok. Hope, it could helps.

You can optimize your sonication condition reducing the cycles number. The optimal range of fragmented DNA is between 200 and 500 bp.  

Hi Yuan,

A 1% agarose gel does not separate fragments below 500 bp well. You will get a much better impression of the size distribution if you run your sample on a 1.5% - 2% gel. Moreover, my impression is that you may have loaded too much material on the gel (the very large U-shape of your bands suggest that you deplete your Ethidium Bromide). Try to not load more than 500 ng - 1 ul of each sample/lane.

Finally 2 things you don't mention: do you de-crosslink your samples before loading them on gel? And are these samples RNAse treated? Both are essential to get a good impression of the quality.

Good luck! Daan

Hi,

I agree with Daan, it looks like over-loading on the gel first, second like degradted RNA. It is very hard to get so small DNA fragments by sonication from fixed chromatin.

Thanks everyone, these suggestions are  really helpful! 

This is addressed to Irina Panteleeva. I am wondering how do you tell these fragments are degraded RNA? Thanks ~

DNA shearing, especially from fixed chromatin has its limits, it is very hard to get a tight distribution below 200 bp for DNA, while RNA gives easy so small fragments. You should include RNases treatment before analysing samples. Be aware that RNAses are inhibited by SDS which is usually included in cell lysis buffer.

Hi Yuan,

The gel looks like you have not reversed the cross-link. After sonication, did you treat the sample with RNase and Proteinase K?  I also suggest to do a cycling gradient that way you can see which cycling condition gives you proper shearing. 

Thanks,Charlene Babra Waryah! I treated the samples in 02M NaCl for 4 hours at 65℃ to reverse the cross-link, followed by RNase and Proteinase K treatment for 1 hour. So I am wondering why the gel always looks like this. Thanks so much for your advice.

Thanks everyone, I have optimaze the sonication conditions, the indexes used to generate fragmented DNA are as follows: 100 Watt, 20 Hz, 10s on, 30s off, 4 cycles; They worked well in my experiment. Hope it will help! Thanks again.