Before post here, I have searched the forum with "sonication" or "Bioruptor", but my woes still exist. I'm using the Bioruptor UCD-200 to sonicate the cultured MCF-7 or other cancer cells for chip-seq. I have tried 30-60 cycles at low power or 15-40 cycles at high power. Although sometimes i got the ideal fragments (200-500bp),the results were very inconsistent. What do you all think of the results that I am getting (see attached images)? I am using the same fixation as well for 1% formaldehyde and then glycine to stop the fixation at RT.The cells were lysated with Cell Lysis buffer (20 mM Tris-HCl pH8, 85mM KCl, 0.5% NP-40, 15min), and followed by Nuclear Lysis buffer (50mM Tris-HCl pH8, 10 mM EDTA, 1%SDS,30 min). As advised in some paper,a snap-frozen step was added before sonication. other conditions were detailed in images. I use the 1.5-mL Eppendorf tube and the concentration is about 5*10^5 cells/120 uL/tube. By the way, according to the Bioruptor manual, I spin down and vortex every 10 cycles. But when spin down, there would be white precipitates at the bottom of the tubes.Is the precipitate SDS? In addition, I just leave very little ice in the tank since it would decrease the sonication efficiency. So, what is the deal?
Thanks a lot for any input.
Edited by jancho.wang, 16 June 2012 - 02:33 AM.