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DNA and histone methylation help


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#1 2bornot2b

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Posted 15 January 2011 - 03:50 PM

Hi there,

I'm new to epigenetic research, and I'm trying to construct an adequate proposal, but unfortunately I seem to be getting stuck on the protocols. Any help would be really appreciated to this newbie.

Firstly, I would like to perform ChIP-Seq on my samples, but I'm not sure if I am able to do this with blood samples as the brochure says 'life cells' are required. What would I need to do?

Secondly, am I able to perform standard DNA isolation and then use formaldehyde to crosslink, or are cells necessary?

Lastly, what protocol would I use in order to isolate DNA with the histones intact, as all the isolation protocols that I've seen use proteinase K, which kind of defeats the point.

I know that these questions may be a bit silly, but I am desperate to find out how to get it right, and with all the information out there, it seems a bit difficult to find exactly what I'm looking for, so I would rather ask the experts who actually use this technique.

Once again, I would really appreciate any sort of help that is offered :)

#2 pcrman

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Posted 15 February 2011 - 07:41 PM

Firstly, I would like to perform ChIP-Seq on my samples, but I'm not sure if I am able to do this with blood samples as the brochure says 'life cells' are required. What would I need to do?
>> I think people have done ChIP assay using blood cells.


Secondly, am I able to perform standard DNA isolation and then use formaldehyde to crosslink, or are cells necessary?
>> you need to crosslink protein and DNA in living cells not after DNA isolation.

Lastly, what protocol would I use in order to isolate DNA with the histones intact, as all the isolation protocols that I've seen use proteinase K, which kind of defeats the point.
>> As stated above, you need to do crosslinking before DNA isolation because DNA isolation will strip DNA of protein.




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