No DNA after sonication for ChIP - (Mar/17/2008 )
I have a big problem whith my chip, after DNA sonication I reverse crosslink to check the smear.
So I reverse crosslink overnight with rnase A, then incubate with proteinase and perform a phenol chloroform extraction and ethanol precipitation. I can see a pellet and according to the nanodrop, there is DNA (4OOng/ul). BUT when run an agrose gel, there is nothing, no smear, no 4kb band, nothing... It's a 1% gel, I can see clearly the ladder.
Two months ago I already performed such experiments and I saw a nice smear, and the protocol is the same!
the only thing which is different are the conditions of fixation. I perfuse the liver 10 minutes whith 1% formaldehyde and then 10 minutes with glycine.
Has anyone an idea about the location of my DNA???
The most likely steps at which DNA is lost are the purification steps. Also make sure your RNase is DNase free.
After reverse crosslink, run a gel and you should be able to see your DNA although remaining protein may interfer migration. Use Qiagen PCR purification column to purify your DNA. The procedure is: after reverse crosslink, and RNase and proteinase treatment, add 5X PB buffer, and following the remaining steps of the qiagen protocol. The column save you both time and your DNA.
The pallet you saw may not necessarily be DNA.
if you change the protocol using MNase instead of sonication, will it help?