how to shear tissue DNA - (Jan/03/2006 )
hi everybody, I just started to do the CHIP assay on rat hippocampus, I also have problems about shearing the chromatin DNA. so I join this forum for help
the following is my procedure.
1: I isolated the hippocampus of rat then minced in a 35mm plate filled 4 ml cold PBS
2: then I add the Formadehyde to a final concentraton of 1%
3: I incubate the plate at 37 C for 15 minutes(because the PBS is cold, so prolonged the incubation time)
4: I isoated the nuclear then lysis it in SDS lysis buffer.
5: and then I sonicate the lysis, the total volume I used is 800 ul.
I tried many sonicte conditions, the power setting range from 20% to 30% (KS 600 sonictor) the total time range from 60sec to 180sec (usually I will sonicate 20sec or 15sec, and then brake for 2min), I must note that there is no foaming when I did it.
but the sheared DNA is not good. there is a bright band at 4KD.
I am very frustrated about it
in addition, I found that the bad shearing will not enfluence the CHIP relults, I mean I can also get a positive band after PCR about 30 cycles (but not in the no antibody control), why this happened?
hope for your help
I'm working on a ChIP protocol for brain tissue as well and know of only only one detailed description of this (tsankova nm et al 2004). They do not mention incubating at 37 while crosslinking. Also after fixing for 15min, did you stop the reaction by adding glycine (final conc. of 0.125M) ?
Thank you for your reply. I did add the glycine to the final concentration of 0.125M.
the following is my procedure now.
1: I isolated the hippocampus of rat then minced quickly in a 35mm plate filled with 4 ml room temperature PBS
2: then I add the Formadehyde to a final concentraton of 0.8%
3: incubate the plate at 37 C for 10 minutes.
4: I add the glycine to the final concentration of 0.125M, incubate for another 5 min at RT.
4: wash the tissue for at least 3 times with PBS (plus protease inhibitor), once with cell lysis buffer.
5: and then I homogenate the tissue in the cell lysis buffer (plus protease inhibitor) to produce crude nuclei.
6: 5000 rpm, 5 min. I discard the supernatant. and add the SDS lysis buffer (plus protease inhibitor) to resolve the nuclei for 20 minutes.
7: add the equivalent CHIP dilution buffer to the SDS lysis buffer.
8: and then I sonicate the sample with 800 ul volume in cone tube.
(KS600 21%output 20sec per time)
the remain step is as the same as others.
attached is my sheared DNA fragment.
1: I found that it is difficult to sonicate the DNA fragment when I used the 1% Formadehyde, I tried many sonicate conditions, but there is no significant change with the sheared DNA fragment. so I had to decrase the concentration of Formadehyde.
2: there is some improvement when I used the low concentration of formadehyde, but there is also a 4Kb band, I do not know how to resolve it.
3: I found that poor sonicate will results in high yield but low sensitivity. and reverse time is another question. It seemed that the short DNA fragment is easy to be detached from the crosslinked protein. long DNA fragment will produced after long incubation. thus overnight reverse will result in high backgound when the sonicate is not so good.
welcome anybody to give me some advise