I need to obtain nuclear and cytoplasmic fractions from my cultured cells (HEK293AAV) but I cannot seem to get pure fractions.

I am using the nuclei EZ kit. I am following the manufacturer's instructions with the only modification that I have scaled down the volumes of reagents used as I am only using the cells from a 24 well plate well instead of a 100mm dish.

To check if my fractionation has been successful I am performing qPCR reactions in both fractions (nuclear and cytoplasmic) with both GAPDH and a mitochondrial gene. This should allow me to assess the degree of contamination from one fraction to the other.

I have tried this several times, even doing the extra-wash of the nuclei pellet suggested in the troubleshooting section, but I am still getting most of the mitochondrial gene detection in the nuclear fraction.

Can you suggest any tips to improve this?

Thanks a lot,

Paula