DNA shearing optimization for ChIP assay - (Mar/22/2006 )

Hi everyone,

I'm a beginner to ChIP assay and no one in my lab has experience of this before. I'm trying to optimize the condition of DNA shearing with the only one sonicator in my lab - Soniprep 150 (A SANYO MSE Ultrasonic Disintegrator ). I checked the length of DNA fragment in 2% agarose gel, but had no idea about how to interpret the result and how to modify the sonication condition next time. So I hope that maybe someone can give me some hints.

The picture of gel has been attached.

Sonication condition: 2s on, 10s off, at power 5 for 5 times with MSE Soniprep 150. No foaming at all during sonication.

Sizes of the DNA ladder were indicated in the attached picture.

Lane 1: before sonication
Lane 2: after sonication


Thanks for your valuable advices!


Jo

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i am a little perplexed with your lane 1. it seems that it has already been "sheared" but lane 2 looks like a typical sample from sonication,

how did you prepare your samples in lanes 1 and 2 before loading onto a gel?

Nick

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Well, both samples in lane 1 and lane 2 came from the same 6-cm dish with a 90% confluency at the day of harvesting. For crosslinking, I added formaldehyde directly into the culture media to a final concentraction of 1%, and incubated the cells at R.T. for 20 min. Glycine was subsequently added (final = 0.125 M), and cells were incubated at R.T. for a further 15 min. After scraping cells in 1 ml cell lysis buffer, cell pellet was left in the tube and resuspended in nuclei lysis buffer. Nuclear extracts were then split into two samples - sample in lane 1 was left untreated, and sample in lane 2 was sonicated. Phenol/chloroform extraction was performed following reverse of the crosslinking after an incubation at 65℃ for 4 hr.

Thank you very much for your patience!!

Jo

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