Hello all. I am trying to do ChIP-seq with testis tissue, and I have been having a lot of trouble with sorting out my shearing conditions. Essentially, my sheared and unsheared chromatin always seems to look the same, and it looks like too small a size for unsheared chromatin, as you can see in the attached photo. I have tried a lot of different sonication times (as listed in the image) and I tried varying fixation times and concentrations of formaldehyde (this image is 1% for 15 min at RT). I think the chromatin looks degraded, but I can't see how that is happening as I collect the tissue into fresh, sterile, ice-cold PBS, briefly break it up by hand with a sterile pestle, and then fix it. Any suggestions or advice would be hugely helpful!
Dear Jocelyn, it does look like some degradation was going on, and I am wondering if you can simply use the DNA for ChIP? Can you take an aliquot of the sample to do an IP and then qPCR to see if the sample is good for your ChIP experiment? Maybe you can have some nuclease inhibitors during the chromatin/DNA preparation?
The DNA concentration in reaction was way to much... Reduce the DNA concentration and you will get good bands.
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