Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

MNase assay - how to quantify DNA

MNase

  • Please log in to reply
3 replies to this topic

#1 yaeldeb

yaeldeb

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 29 September 2014 - 02:57 AM

Hi,

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.

Yael 



#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,504 posts
252
Excellent

Posted 29 September 2014 - 06:31 AM

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.



#3 yaeldeb

yaeldeb

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 30 September 2014 - 02:36 AM

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.

Thanks



#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,504 posts
252
Excellent

Posted 30 September 2014 - 04:16 AM

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.







Also tagged with one or more of these keywords: MNase

Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.