3 replies to this topic

[mikew]

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?

[paulina]

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.

Quote

For testing sonication condition, I think you don't need to purify the DNA after reverse crosslinking and before running a gel.

I am sorry, this is not true. after crosslinking, do a phenol/chloroform extraction and take 20 ul to run a gel.

Edited by pcrman, 16 March 2005 - 11:49 PM.

[mikew]

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.

[badcell]

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!