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Formaldehyde cross linking problem!!! - (Feb/09/2012 )


I am trying to optimise ChIP protocol and not able to go pass first step of cross linking!

I am collecting 3-4g plant tissue and crush it in liquid nitrogen. Followed by either storing them in -80 or use it to corsslink in a buffer containing 1% formaldehyde.

I have tried 1% formaldehyde to corsslink my samples (2.5, 5 and 10 minutes). Interestingly, I do see DNA when I De-crosslink but in (IP) control samples which are not de-cross link I dont see even a faint DNA band. Controls without formaldehyde gives good band in de-crosslink or non de-crosslink. This has led me to understand that the samples are over corsslinkned.

1) I am wondering if any one has done cross linking with samples which are first powdered in liquid nitrogen?

2) is 1% formaldehyde ( 37% formaldehyde containing methanol is used) concentration too much for powdered sample?

Cheers for any help in advance!


Hi Chip,

From the information you gave, it sounds like on an agarose gel with a non-decrosslinked sample you do not see DNA smear. That is probably because your DNA is bound to protein and won't migrate down the gel. Also, my protocol calls for de-crosslinking of the input control sample, just like the IP'ed samples. Additionally, what are your shearing conditions? My understanding is that you simultaneously optimize cross-linking and shearing because the two go hand in hand.

I hope this is helpful...


Hi Chip,

Any luck?



Hi Emily,

Thanks for your info. I was trying to use plant material powdered in liquid N2 for cross linking and nuclei isolation together in one step.

I came across a paper ( which shows nicely -DC and +DC gel picture to optimise FA crosslinking step. I was expecting a faint band of DNA on gel which might confirm that samples are not over cross-linked.

The sonication worked well - I did the pull down with controls and got no signal on qubit! It looks that as my samples were over crosslinked I might have over sonicationed to achieve right size DNA smear and in process lost my Ab epitope :-(

I have now moved on to conventional way of crosslinking i.e. infiltration followed by crushing sample in liquid N2 to isolate nuclei which seems to be working well : -) and now I am at DNA pull down step and trying to troubleshoot some problems!

Thanks again.