Hi Hani,

I think it is very likely that this an artifact. In most ChIP experiment, the DNA concentration of the input is 10 - 1000 fold higher than in the IP'd samples. A PCR that runs well in the input, may therefore be prone to generate all kinds of non-specific / unwanted products in the IP'd sample. Besides running your melting curve, I also advise you to run your PCR product on a 2% agarose gel to get a precise idea of the specificity of your product (presence of other bands) and the size difference.

Considering that you did a melting curve, I assume this is a ChIP-qPCR experiment. 2 possible things I can think of:

- Was your amplicon very small? Could it be that instead of a specific product, you now are generating (and sequencing) mainly primer dimers?

- Does your target sequence have repetitive characteristics? Could it be that instead of your specific sequence, you are now amplifying an abundant, related, repeat sequence?

Good luck!

Hi Daan,

Thank you for your anwer. Actually it can not be an artifact because it is in all inputs for all samples and its is missing from all IPs from all samples and it is exactly the same 15 nucleotide. The melting curve have shifted 1.5 degrees only because of the missing 15 nucleotides. The amplicon is normal for any promoter primer and it can not be a primer dimer as the peaks are for a real product.

It is very strange what we are seeing, thats why any input would be helpful in that regard

Hi Hani,

It's difficult to guess from far away what it can be. One control that you should do, is dilute your input to the same concentration as your IP'd sample (pr dilute it to a concentration where the Ct in your qPCR should be about the same). If low DNA concentration causes this effect, it should now also show up in your input.