I need some help

Hello,I know there are scientists among the members of this forum and some science-oriented people too, so maybe you can help me.I need to find (and buy) and enzyme or reagent that DEmethylates genomic DNA in vitro. That is, I have extracted genomic DNA from my organism into an extraction buffer (Tris and EDTA probably), and now I need to DEmethylate it (cytosine methylation, that is). Some companies sell 5-Azacytodine, which is a methyltransferase inhibitor. That's obviously not good for me because it has to be added to the organism to produce hypomethylation in vivo.
Any ideas on what could possibly do the job?

Thread moved here from the Proposed New Topics forum.

As far as I am aware there is nothing readily available that will do this in terms of demethylating the existing DNA.What is it you want to do with the unmethylated DNA exactly? Is it a control for some methylation assay, i.e. bisulfite sequencing or MS-PCR? If you just want demethylated sequence you could just whole genome amplify it and there would be little chance of pulling out a methylated sequence. You could even add a step using a methyl-Cytidine antibody to literally pull the methylated DNA out, but that would probably be unnecessary.Alternatively is there any suitable cell line you could get the DNA from, or a suitable tissue for primary cell culture? Then you could do a treatment with Valproate or 5-azacytidine and extract your genomic DNA from the cell culture.TTFN,WK

I did a form of MSAP on transposons - it's called Transposon Methylation Display (TMD). I cut the genome with methylation sensitive enzymes HpaII and MspI, ligate adaptors and PCR with an adaptor primer and a transposon primer. That way I get random fragments that show me the overall methylation status near transposons (I compare different generations of newly formed allopolyploid wheat). However, a lack of amplification means that the DNA is either methylated or that the sequence was eliminated. Doing the same reaction with hypomethylated DNA can give an indication of which is true. Whole genome amplification on wheat? Is that possible? Hexaplods of wheat have 17Gb genomes, only a tiny fraction of which is sequenced, which is annoying considering this is both a model organism for research and probably the most important agricultural crop in the world! We are going to try cell cultures for a different reason. But I think 5-Aza will not be good because transposons have a tendency to go crazy when the genome is hypomethylated in the plant. That's why I want to do it in vitro.This whole methylation business is tricky...
But at least I have an article from it, LOL! (I'm the second one)

Well since you didn't say it was in wheat I couldn't really be expected to know that was the organism you were talking about. That said I don't see why it shouldn't be possible, it isn't like you are doing gigabase length PCRs.I don't see why 100ng of wheat genomic DNA would be any less suitable than 100ng of any other sort of genomic DNA for WGA, and a quick slow and laborious back of the envelope calculation suggests that 100ng of genomic DNA should still have at least several thousand copies of your entire genome present even for a 17Gbp whopper. How big are the fragments you get usually? I guess that could be a problem if the WGA process wouldn't produce a long enough fragment.TTFN,WK

Sorry, sometimes I forget where my DNA comes from... I have never done WGA...The Qiagen website says the fragments from WGA are 10-100kb, that's OK by me.I think I'll check the option with my advisor, Thanks!