A single band after sonication? - (Aug/27/2008 )
I got a problem with sonication in preparation of ChIP assay
I start out with some mouse liver frozen tissue
I thraw them on ice, cut them into small pieces by razor blade and homogenize them with a glass homogenizer in solution with sucrose and protease inhibitor (supposed to leave the nucleus intact)
Then I centrifuged the sample at 4C 2400g 10min to get a pellet, wash it with ice cold PBS and centrifuge again
The pellet is then resuspended in upstate ChIP assay kit SDS lysis buffer (100mg tissue -> 1.2ml buffer)
I split them into 300ul each and sonicate with 4 different conditions
mosonix 3000 with microtip, output 1.5, 10sec on, 20sec off, X4, X6, X8, X10 times
Then I did NOT decrosslink the DNA and went ahead to run a 1.5% agarose gel with 1X TAE
I couldn't see a clear smear whatsoever, and there is a quite discrete band at about 500-750bp
I tried to treat the sample with RNase A for 30mins and run the gel again, the band is still there
Did I do something wrong??
can you post a picture?
I saw KPDE wrote:
Reversal of crosslinking is very important for looking at your fragment sizes on agarose. You will mostly get high molecular weight smears (sometimes the DNA will remain in the well) unless you reverse the crosslinks.
And it seems right, as yesterday when I saw that strange gel, I did decrosslinking by NaCl 65C overnight and phenol chloroform
Now I see the smear and the band at around 500bp is gone
So, just for interest (and in case you wonder): I TRIED TO RUN GEL TO CHECK DNA SIZE WITHOUT DECROSSLINKING AND IT DOESN'T WORK!!
the next problem now is the smear I got is from 1kb to 5kb
The most vigorous condition I used is output 2.0, on/off 10/20s, and 10 strokes
Have anyone tried sonicating DNA from mouse liver??
Experience and Suggestions will be more than welcome