MNase assay - how to quantify DNA - (Sep/29/2014 )
I'm trying to set up a simple MNase assay where I am compare global chromatin compaction of two melanoma cell lines. I want to make sure that I have the same amounts of DNA before I add the MNase. One protocol I have suggests counting nuclei. One suggests adding 2M NaCl to the nuclei and OD another suggests adding 1M NaOH to the nuclei and OD.
I did a pilot where I took ODs with either NaCl or NaOH and then took the nuclei sonicated them and did phenol chloroform and then OD.
The reading after phenol chlor was 500x more than the reading before.
Can anyone recommend an accurate way of measuring the DNA? Or even point me to some simple MNase protocols.
Phenol absorbs in the UV, so any phenol contamination will look like DNA when measured with UV absorbance. You probably need to remove the phenol in any case. Try a chloroform extraction (or more than one) to remove phenol.
I did use chloroform after the phenol but ideally I want to measure the DNA before the phenol chloroform.
Here is the basic protocol.
I take two tissue culture lines, I lyse the cell and spin down the nuclei and then I want to aliquot equal amounts of DNA from each cell line. This is where I am stuck.
Once I have the equal amounts aliquoted then I want to do the MNase, RNAse A, proteinase K and phenol chloroform.
If you still have nuclei present in the sample untreated with phenol, then the DNA may not be accessible for measurement. Is the exact amount of DNA present before treatment important? Perhaps you can normalize DNA amounts after phenol extraction. If it is important, then perhaps you could count nuclei with a FACS machine.