HELP with preparing DNA for plant ChIP seq

[sally adams]

Hi everyone,

I am currently trying to prepare samples for ChIP seq analysis but keep loosing all my DNA in the last purification step. I am using an antibody against a transcription factor and get good enrichment of targets (tested by qPCR). The only problem is I never seem to be able to get sufficient DNA back out off the qiagen column to test the concentration using the Qubit system. I have double checked that the DNA elutes from the protein A beads and if I do exactly the same process with input DNA (which I have diluted to the same concentration as my ChIP samples) I am able to easily purify the DNA using these columns. I have checked the pH of my samples (they are about pH 7.5 as recommended). The only thing I can think of is that the DNA is not being reverse crosslinked in my ChIP samples (I add 5M NaCl and heat overnight at 65C). I was wondering if anyone has had a similar experience or could give me any suggestions. It is slowly driving me nuts and any help will be very gratefully received.

Thanks Sally

[Chipper]

Hi,

do you see the same enrichment levels with qPCR before and after the minelute (?) columns? In that case you are likely just overestimating the DNA conc before column purification..

[sally adams]

Hi,

Yes, I am using the minelute columns (sorry should have mentioned that). I don't seem to be able to test my elutes prior to the minelute purification step as the qPCR seems to fail. I am guessing this could be due to the high level of SDS I have in there. However, I do get similar enrichment levels in the qPCR with my minelute cleaned samples as when I use the Chelex purification/reverse crosslinking method. I am beginning to suspect that the level of DNA I am getting back is just far too low a concentration to be measured by the qubit. I get decent readings with the nanospec though...