We are interested in ChIP for transcription factors. We would like to know the ideal fragment size for ChIP for murine bone marrow cells/ 5Fu progenitors. We also require conditions i.e. Power, Duty Factor, Burst, Duration, No. of cycles - which would be ideal for Covaris sonicator. We are currently using 10 million cells in 1ml RIPA buffer in milliTUBE 1ml AFA fiber (100)-tubes. The results for our current efforts are attached as a figure with the conditions we have used. So far our fragment size is quite dispersed i.e. from 100bp to almost 3kb.
Hi,
Your samples look like RNA being below the 100 bp mark... Do you RNAse treat? If so, for how long? You should also include a non-sonicated sample to show that the previous steps do not damage the DNA and as a control for RNA.
Also, when I look at the top of the gel, there seems to be a small band up there. That, to me, looks like the genomic DNA. Could you please provide the steps that you do to purify the DNA? I think that will help with providing the most meaningful answers.
Hi Nicholas,
thank you for helping us. The idea of running unsonicated DNA is a good one. we will try it.
We put 10^7 BM cells in 1ml RIPA buffer. We incubate on ice for 20 mins and proceed with sonication. Post soincation, we use 5% of sheared DNA (50ul) for RNAse treatment for 30 mins at 37 degrees and subsequently perform proteinaseK treatment for 2 hrs at 65 degrees. From this step onward we have tried two approaches- a)loading the DNA straight away on a gel b)column purifying it and then loading it on a gel. In both cases, the 100bp bands are still visible, though the coloumn purification gets rid of the genomic DNA that is otherwise visible on the top of the gel. Does this answer your question?
Hi Shiva,
Did you ever figure out sonication conditions? We've been trying to shear chromatin using the covaris recommended buffer (not RIPA) for DLBCL lines. We use 20 million cells per ml in the 1ml militubes with AFA fiber. We usually cross-link our cells in 1% formaldehyde for 10 minutes. Wash the cells with cold PBS (3X), resuspend the cells in the covaris shearing buffer (to 20 million per ml), incubate on ice for 20-30 minutes and then start the shearing. Our sonication conditions are more or less around the following parameters: 140W, 5% ADF, 200cycles per burst, 20 minutes. We've had varying success with the shearing as it seems that the results vary between runs.
As a side note: for TF CHIPs wouldn't you want fragment sizes between 200 and 500 bp? Fragments that are too small (100bp) would not have your tf bound to them after such an intense shearing.
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