My ChIP protocol with sonication alone gave me good enrichment of my GOI, but made me concerned b/c the DNA fragment sizes were 5+kb at times. Therefore, I added a MNase step (still with sonication) and my DNA fragment size is now
I've only had experience with MNase only. But from what I've read Alot of things can effect sonication alone such as keeping your samples on Ice which can drastically effect sonication efficiency. Now in my protocol I have a very brief period that I sonicate my samples briefly after MNase digestion. This is to only help rupture the nuclear membrane and gain access to the chromatin.
Hello Matt,
If your sonicator supports metal rods for direct treatment of the solution (you dip it into your sample) - it can give better effect. For example, using one sonicator this way I could achieve 1000 bp lenght, then we bought newer machine and achieved 300 bp. If you use formaldehyde for cross-linking, you can decrease its concentration for better shearing of chromatin. As for MNase, I haven't worked with it.
When you say no enrichment do you refer to how much DNA you recover by mass or do you perform qPCR on specific loci with no difference between your sample and control?
In theory small fragment size will give you better resolution and it doesn’t have to affect the enrichment which is determined by the fraction of available epitopes versus the concentration of your antibody. You are not going to pull down more 0.3kb fragments than 5 kb fragments. Intuitively, if you pull down 1000 fragments of 5Kb and 1000 fragments of 0.3Kb then you will have more DNA by mass from the 5Kb sample but this is not how the enrichment is tested. The enrichment is calculated by normalizing to input.
I wouldn’t use MNase in this case but try to optimize sonication. With normal sonicators you can achieve a distribution in the range 200bp-500bp more or less. It is better not to go down.
Technically, we perform MNAse digestion to avoid any false positives, so even if you find a decrease in target enrichment its fine. In my case, I perform HAP purification after MNase digestion to ensure only mono-, di- and any tri- nucleosomal length DNA with maximum enrichment of mononuleosomal DNA. An article on this method may be handy to you.
http://www.ncbi.nlm.nih.gov/pubmed/22183596
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