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#39194 Help!! Jammed at DNA shearing

Posted by s2289 on 08 October 2009 - 02:14 AM in ChIP and Next Generation Sequencing

Hi guys, I really need your help.

I am performing ChIP recently, an odd band always showed up when I ran the agarose gel to check the size of sheared DNA.

Briefly, formaldehyde is added into cells at a final conc. of 1%, for crosslinking for 10 min, and then the reaction is stopped by adding glycine (final conc. 0.125M) for 5min. Cells are washed and lysed by SDS buffer (+protease inhibitor) to get the nuclei. After pelleting, the nuclei are resuspended in IP buffer (+protease inhibitor) and then sonicated. Sonicator S-4000 is used, and protocol is set to pulse on for 1s and then off for 5s with amplitude of 30, for 50 cycles.

2 kinds of cells had been tried, including thyroid cancer cells ARO, and Head&Neck cancer cells FADU.

The 1st (ARO) and 2nd (FADU) figures show the results from same treatment of 2 cell lines.

I thought the unwanted debris present in IP buffer might cause it, so I centrifuged the sample after the 5th sonication, then ran the gel (3rd fig.), however, the band became sharper and brighter.

Could anyone tell me what happened, and how to overcome??

Attached Thumbnails

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