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Help!! Jammed at DNA shearing

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#1 s2289



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Posted 08 October 2009 - 02:14 AM

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??

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