Hi, I would like to ask if MNase treatment affects the NanoDrop's...

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Wei Chen
Jul 4, 2022
protocol Protocol: Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei
Hi, I would like to ask if MNase treatment affects the NanoDrop's concentration measured by absorbance? I extracted DNA with the same amount of cells, however, the concentration almost doubled after MNase treatment. Should I load the samples at the same concentration or the same amount of Read more Read less more less
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Jordi Moreno-Romero Answered Jul 12, 2022

Centre for Research in Agricultural Genomics

Hi Chen,

I did not know that MNase digestion could affect that much to the absorbance.

Anyway, when you perform different digestion times or MNase concentration you will start with the same amount of nuclei/chromatin, and this is independent of DNA quantification before or after. As you load the same volume of sample on the agarose gel, you should load the same amount of DNA. To have double the amount of absorbance after MNase should not affect the fact that you start with the same amount of nuclei/chromatin, although the initial concentration would be underestimated.

I prefer to measure DNA concentration with the absorbance instead of cell number because plant cells often go to the process of endoreduplication, that affects the ploidy level of different cells. Therefore, if the treatment or mutant you want to compare affects endoreduplication, this will affect to the starting DNA concentration between treatments/mutants if you start with the same amount of cells/tissue.

Let me know if the answer helped you to solve your doubts or if my explanation was not clear enought.

Best,
Jordi M-R
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