Sonicator Optimization Trouble - Sonicator Optimization for ChIP-Seq (Mar/23/2011 )
Hello Everyone,
I am currently working on optimizing the sonication conditions for ChIP-seq. Ultimately we are planning to start with mouse spinal cord tissue for the ChIP, but for the purposes of sonicator optimization we have been using mouse brain and liver tissue as controls in addition to the spinal cord. We have used two different protocols. One is from the invitrogen Magnify ChIP Kit that we have purchased and used. The other is essentially the same protocol but it uses different buffers that we made ourselves using information from a previously published paper. What do you all think of the results that I am getting. Please let me know if you need more information about the protocols used in order to evaluate the results. The first image from 3/18 is the one we got using the invitrogen protocol and the second from 3/22 is the one we got using the buffers from the other protocol. In both images the three lanes on the left were sonicated for 1h using a bioruptor UCD-200 sonicator at high power (30s on 30s off) adding ice to the water bath after every 10 min. The three lanes on the right were sonicated for only 0.5h on high power (30s on 30s off) changing the ice every 10min. Within each set of three, the brain tissue is on the left, the liver is in the middle, and the spinal cord is on the right. The desired fragment range is between 100-300bp or 200-500bp. Do you think we are close or do we still have a ways to go?
It appears to me the liver tissue in the middle lane of the first image was best sonicated. A lot of DNA from the other two types of tissues got stuck in the wells making it hard to tell whether the conditions were optimized.
pcrman on Thu Mar 24 05:12:13 2011 said:
It appears to me the liver tissue in the middle lane of the first image was best sonicated. A lot of DNA from the other two types of tissues got stuck in the wells making it hard to tell whether the conditions were optimized.
Thank you for your response. Is the DNA from the other tissue types stuck in the well because they were not sheared into small enough fragments to be able to translocate through the agarose? Is it possible that longer sonication is needed for those two tissue types? From the information I have gotten from the protocols and information I have gotten from this website, it seems that 1hour sonication is already a lot more than anyone else is doing. What do you think?
I am having the same issue as you do! my samples are human cells, I just couldn't get them sonicated. They always stuck in the well. Have you figured out how to solve it? it'll be really appreciated if you can offer me some advice!!!
Just a thought here... DNA could be stuck to the wells due to incomplete de-crosslinking. The bound proteins block movement through the agarose gel. You could try running the gel for longer to see if DNA will leave the wells. Also, maybe try your samples without de-crosslinking them for comparison (as a control). DNA from non-decrosslinked samples should remain in the wells (at least to a much higher degree than decrosslinked).