I have recently initiated my ChIP assay and am currently in the process of optimizing the conditions for chromatin shearing using sonication. The extraction is from chickpea leaves for a total of 3 grams divided into 3 falcons.

For nuclei isolation, I employed a 25 ml buffer composition, as follows: 0.25 M sucrose, 15 mM PIPES (pH 6.8), 5 mM MgCl2, 60 mM KCl, 15 mM NaCl, 1 mM CaCl2, 0.9% Triton X-100, 1 mM PMSF, and 2.5 tablets of protease inhibitor (PI).

Additionally, for nuclei lysis, I used 2 ml of a buffer consisting of 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% SDS, 0.1% sodium deoxycholate, and 1% Triton X-100, along with half a tablet of PI.

I then performed the shearing process using two types of sonicators, and I have attached a picture that provides all the pertinent details about the sonication.

Before the gel loading I centrifuged to pellet debris, and collect the supernatant.

I loaded 5ul of sample

I am currently facing an issue with material presence on the wells and the observation of a smear or very faint band in the non-sheared sample. I would greatly appreciate any suggestions or guidance you can offer regarding this matter.

Thank you in advance for your assistance.