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Formaldehyde cross linking problem!!!

formaldehyde crosslinking cross linking

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3 replies to this topic

#1 chip

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Posted 09 February 2012 - 04:38 AM

Hi,

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!

#2 emilyindallas

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Posted 09 February 2012 - 08:15 AM

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...

#3 emilyindallas

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Posted 23 February 2012 - 09:11 AM

Hi Chip,

Any luck?

Emily

#4 chip

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Posted 14 May 2012 - 05:50 AM

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 (http://www.plantmeth.../content/3/1/11) 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.





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