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DNase not cutting in assay - (Nov/23/2007 )

Hi all,

I'm having some trouble getting my DNAse hypersensitive assay to work... It's simply not cutting angry.gif
here's what I do:
harvest 1x10^7 cells, wash with PBS, lyse in RSB (10mM pH7.5 tris, 10mM NaCl, 3mM MgCl2) plus NP-40, homogenize with down
pellet and wash 3 times with cold RSB, then the assay:
0.1U-2U of DNase in 500uL of nuclei incubated in 37 oC for 25 min... Stoping the reaction with 1% SDS/0.6M NaCl/20mM Tris/10mM EDTA (2h 37 oC)
Phenol/Chloroform followed by EtOH ppt, then run an overnight gel to check the digestion...
and as I said earlier, I'm simply not getting the smear I expect from the digest. I do get chromosomal DNA bands near the top of the gel...

I've tried overloading with 10U of DNAse and digesting for an hour... washing pellet more...

I'm sure my cells are lysing b/c I checked with trypan-blue after NP-40 step...

Is there any way at all that I could be losing digested DNA at the phenol/chloroform/EtOH ppt step? (I hate doing phenol/chloroform/EtOH ppt cuz 99% of the time I don't see a pellet and I feel like I'm working with an empty tube...)


Add 1 ul of Novagen Pellet Paint, and you will be able to see your pellet. Use NF pellet paint if you will use fluorescent detection later with that DNA.