ChIP sonication woes
Posted 17 November 2004 - 01:30 PM
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!
Posted 17 November 2004 - 05:28 PM
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.
Edited by pcrman, 16 March 2005 - 11:49 PM.
Posted 18 November 2004 - 01:36 PM
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
Posted 13 December 2004 - 08:24 AM