Hello everybody,
I'm running the Agilent ChIP-on-chip system. Now I encountered a problem about the sonication step. After purification the sonicated DNA showed a two part pattern (the attached .jpg file shows this pattern): the bigger one is more than 2Kb, the smaller one is averaged 250bp, and there is no wanted part (500bp averaged). When I added more sonicaiton cycles the bigger part diminished, the smaller part increased, but there is still no wanted part (500bp averaged). Has anybody encountered the same problem with me?

I'm using the bioruptor for sonication: intensity: High; cycle: 15s on, 30s off. The crosslinking (0.5% formaldehyde room time 20min) and sonicating condition has been used for our mES cells, it works well indeed. This time I only changed a cell line (MEF cells).

Someone has suggested that I sonicate at Middle intensity first, then turn to High intensity. Has anybody used this method?

So for this kind of cells, how can I modify my sonicating condition or crosslinking condition?
I’m looking forward to your experienced suggestion about this problem.

Thank you all!
Best regards
Randolph

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Hi,
I am using the Agilent chip-chip system too. We have done successful experiments with sonicated DNA the same size as yours, so I wouldn't stress too much about trying to get the 500bp that Agilent suggest.
Clare

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Thank you Clare, but I don't konw if this kind of fragment pattern will affect the following antibody enrichment step. The big one may not be efficiently enriched.

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What antibody are you using?

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The antibody for Oct4, Sox2, Klf4 and c-Myc from Santa cruz. So Clare, have you ever encountered this kind of pattern? I encountered this when I sonicated MEF and iPS cell, but not ES cell.

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I have to change my sonication conditions for different cells. I am doing chip-chip with cell lines and also patient material. I stick to the same cell concentration but have to vary the time (usually 5-7 minutes). I suggest you do a little sonication experiment. Sonicate at 1 million cells/30ul, high, 30sec on/off. And do a few time points....see what works best for your cells On the first day of chip I always do a quick reverse cross-link (usuing about 1 million cells) to make sure my sonication is ok.
Good luck
Clare

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