Good morning colleagues,

I want to share with you my results, hoping that someone could help me to find a possible explanation.

In the picture you can notice a 1% agarose gel where I run PCR amplicons of a gene promoter. In detail, I started from a best prognosis (left) and a worst prognosis (right) primary cell line, I fixed them with formaldehyde, lysed, extracted the DNA and performed chromatin enzymatic shearing with micrococcal nuclease; then I immunoprecipitated it with histone marks indicated in the lower part of the picture. Ultimately, I designed PCR primers to detect the presence of the amplicon in the immunoprecipitated chromatin.

As you can notice, I can observe the amplicon bands (almost 250bp), but in some histone marks IP I can also see higher bp bands which are difficult to interpretate. I exclude an aspecific amplification, as it doesn't happen in all IP bands and the primers I used should be specific for my region of interest.

Do you have any suggestion or interpretation? Have you ever encountered such a problem in your experience?

Thank you in advance

Jacopo