Is this sonication result good for ChIP? - (Jul/31/2007 )

Dear all,

I started to do ChIP. And I am optimizing sonication. Here is the result. The first two lanes are from 10 cycles and the last two lanes are from 15 cycles. Is the result good enough for ChIP?

I really appreciate your advice and comments.

Thanks a lot!

Stormlina

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at first, some advice with pictures attaching : i would suggest to post picture that will size 1000x1000. Your is quite long to download from my small connection
well i don't see precisely the bands of the ladder, but with what i can see from the lanes, i would go for 15 cycles.
Your smear should range from 200 til 700 to be quite safe.

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I've also recently started doing ChIP myself... Im curious about how much you are loading per lane. The experiments I'm doing only use 1x10^7 cells per sonication; so I have to load TONS of the sample DNA (after EtOH cleanup) to see anything on my agarose gels. How much sample or DNA are you running in those lanes?

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I could see clearly the molecular weight markers, thus it is hard to judge whether the sonication is sufficient.

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Hi,

I use around one million cells. I load 10 (the first and third lane) and 20 ul (the second and fourth lane) from around 300 ul extraction solution.

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Hi,

I am sorry about the inconvenient. The range of the smear is around from 200bp to 700bp. Since this is the first time I tried ChIP, I am not sure whether this is ok.

Thanks.

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200bp-700bp is fine unless you are going for single nucleosome resolution. I'd agree with Fred33 that you should use 15 cycles since there are fewer residual high MW fragments.

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