ChIP sonication woes - Losing DNA? (Nov/17/2004 )
I am trying to opitimeize sonication conditions for my ChIP.
Even at 1x15 secs at low power my DNA is at ~100-300 bp.
My cells that I cross link, but do not sonicate give me a small
smear at ~50 bp, and two bands at 1kb and 2.4 kb. This may be RNA.
I think I am losing most of my DNA, but I do not know how.
After I sonicate, I reverse crosslink at 65 degrees for 4 hours, then proteinase K
followed by phenol/chlor and precipitation.
I have no idea what is going wrong!
Any theories?
That's weird.
Are you sure you didn't lose your DNA during purification? How many cells did you use? For testing sonication condition, I think you don't need to purify the DNA after reverse crosslinking and before running a gel. A setting at 1x15 secs at low power should not break your DNA into 100-300 bp.
I am sorry, this is not true. after crosslinking, do a phenol/chloroform extraction and take 20 ul to run a gel.
Thanks Paulina.
I use 1-2 million cells. You say that I don't need to purify the
DNA after reverse crosslinking (4 hours 65 degrees with ~200 mM NaCl)?
So, I can simply take a portion of the lysate and run this on a gel?
This would be great as I suspect that am losing the DNA somehow during
purification.
I believe that it is necessary to revert the crosslinking and purify the DNA after sonication to check the fragments size- In case you don't, you will get an electrophoretic mobility shift to higher sizes due to the increased weight of the crosslinked proteins. 10x6 cells is not too much. I also work with low number of cells and usually add 20 ug glycogen to the DNA precipitation mix after phenol:chloroform. Hope it helps!
Hi,
I have a similar problem-if I reverse cross-linking and phenol chloroform extract samples that were cross-linked with formaldehyde but not sonicated I can detect 1500-200bp fragments on agarose gel. I'm not sure why I'm shearing the DNA before actually sonicated. Any ideas on this?