Help for sonication - (Dec/20/2006 )
Hi everybody!
Is it possible that somebody helps me for evaluate my sonication? The three lanes are same sonication but different cells.
My protocol: 1) Cells fixed with 1% formaldehyde during 10 min at RT (I have added formaldehyde in the culture cells medium which should be at 37°C,
2) Stop of fixation with glycine during 5 min at RT
3) Wash of cells 2X with PBS+ protease inhibitor
4) Srape cells in 4ml PBS and centrifuge during 10min 4°C at 4000rpm
5) Resuspend nuclei in 2ml of nuclear lysis buffer+ PMSF
6) Sonicate DNA with Branson sonifier B15 (7X6pulses with 1min between each 6pulses)
7) Spin lysate at 14000 rpm 4°C 10min
8) Take an aliquot for evaluate DNA shearing
9) Proteinase K and RNAse A during 1h at 37°C
10) Phenol/chloroform/IAA
Thanks in advance.
Is it possible that somebody helps me for evaluate my sonication? The three lanes are same sonication but different cells.
My protocol: 1) Cells fixed with 1% formaldehyde during 10 min at RT (I have added formaldehyde in the culture cells medium which should be at 37°C,
2) Stop of fixation with glycine during 5 min at RT
3) Wash of cells 2X with PBS+ protease inhibitor
4) Srape cells in 4ml PBS and centrifuge during 10min 4°C at 4000rpm
5) Resuspend nuclei in 2ml of nuclear lysis buffer+ PMSF
6) Sonicate DNA with Branson sonifier B15 (7X6pulses with 1min between each 6pulses)
7) Spin lysate at 14000 rpm 4°C 10min
8) Take an aliquot for evaluate DNA shearing
9) Proteinase K and RNAse A during 1h at 37°C
10) Phenol/chloroform/IAA
Thanks in advance.
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What, in particular, were you concerned about?
Hi KPDE!
I would want to know if my sonication is good to do a ChIP with specific antibodies.
I think that I have some problems with my sonication because at the end of the last ChIP, I have had many background with no enrichissment in my experiment with antibody (I have done many washes, blocked my beads).
I would want to know if my sonication is good to do a ChIP with specific antibodies.
I think that I have some problems with my sonication because at the end of the last ChIP, I have had many background with no enrichissment in my experiment with antibody (I have done many washes, blocked my beads).
It looks like the majority of your DNA is smaller than 1kb, which is acceptable for most people.
I'm not sure why insufficient sonication would affect your background. In our experience, when sonication is insufficient, either the amount of soluble chromatin is so little that we don't get any PCR signal for the IP or we can't pull any of it down (and again, don't get any PCR signal). If you are having background problems, there are a number of things to try. The first thing we usually do is titrate the amount of chromatin used in each IP (I usually start with 4X dilutions) until we find the dilution for optimal enrichment. This is because the non-specific binding which leads to background is much more concentration dependent than the specific binding to the antibody (i.e. decreasing the chromatin concentration decreases the background signal faster then the specific signal). Anyways, good luck.
Joel