ChIP DNA yield problem - (Jan/09/2007 )

ive been trying recently to optimise my sonication for ChIP, but im firstly having problems with obtaining enough DNA from samples.
im working from chopped up frozen tissue (mouse brain), and i cross link 10 mins RT 1% formaldehyde, add glycine to 0.125M final leave for 5 mins, wash x2 in PBS, homogenise in a generic lysis buffer and sonicate at a wide range of settings and spin down to obtain supernatent. i add RNase and decrosslink overnight at 65 degrees. precipitate with 100% etoh, prot K digestion after that and then i've tried both PC and columns to cleam up. either way i get small (~20ng/ul) yield. after crosslinking i have a small amount of transparent 'gloop' in the supernatent that im sure isnt meant to be there.
the only thing i can think of is that the DNA crashes out of solution at some point for asome reason - any ideas?
thanks

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brains are lipid rich, could the "gloop" be that?

also I think there are special methods of isolating DNA from brains, maybe emply these to isolate your DNA after ChIP.

Nick

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If you're NOT using a column specially designed for lipid rich tissues then the lipid portion of the brain tissue will clog your column, leaving most of the DNA within the column. However, Phenol:chloroform extraction will remove all lipids within the sample and has proved very reliable for me when isolating RNA (adding Trizol for this) for rtPCR.

At any rate, does anyone else have some input?

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