Advice on sonication (c2c12)
Posted 19 April 2012 - 01:59 AM
I am still having a few issues with sonication and really hope someone can help me out this time
I am sonicating C2C12 cells with a Sonics Vibracell using a 3mm stepped microtip, in approx 500ul. I'm using 25% amplitude, 20 sec on/30 sec off. The problem is no matter how much I sonicate, I can't seem to shear the chromatin any smaller than 5kb. I've tried different crosslinking times, different power settings, etc and nothing seems to make a difference.
For example (see attached pic):
Lane 1 - 100bp marker
Lane 2 - 15' crosslinking at RT, 15x20s bursts
Lane 3 - 15' crosslinking at RT, 20x20s bursts
Lane 4 - 30' crosslinking at RT, 15x20s bursts
Lane 5 - 30' crosslinking at RT, 20x20s bursts
Lane 6 - 15' crosslinking at 37C, 15x20s bursts
Lane 7 - 15' crosslinking at 37C, 20x20s bursts
Lane 8 - 1kb marker
All samples have been RC, PNK and RnaseA treated. The reason for the crosslinking times is that I was originally doing 30 minutes at 37 degrees (someone else's protocol) and thought this may have been the problem, but cutting the time and temperature back doesn't make a difference.
The other odd thing is that if you look closely, you can see the nucleosomal ladder (I have not seen this happen to anyone else with sonication) and it seems that I am shearing most material down to a single nucleosome.
Any suggestions would be greatly appreciated, I have been trying to figure this out for months!
Posted 21 April 2012 - 02:42 PM
Posted 23 April 2012 - 10:16 AM
also, fifteen 20sec pulses seems a lot to me.......
Edited by chabraha, 23 April 2012 - 10:17 AM.
Posted 30 April 2012 - 01:35 AM
Ultimately I want to do ChIPSeq, so I need a size range of 100-300bp approx. Do you think this is going to be a problem based on how my DNA is shearing?
@chabraha - I am already giving my sample a good mix during sonicating, but thanks for the tip I am sonicating approx 15x10^6 cells in 500ul.
Posted 30 April 2012 - 01:38 AM
and uncrosslinked C2C12 cells (5x20, 10x20 and 15x20).
Just to show what I was trying to say.
Posted 30 April 2012 - 07:29 AM
Are you crosslinking C2C12s after they for myotubes? This could certainly give you a harder time with sonication. Last month I was trying to do ChIP on skeletal muscle and that was a pain. I gave up on trying to get any significant amount of small fragments.
I think you should be fine for ChIP-seq since you have a significant amount of material in the 100-300 range. Also, running your input control in parallel will remove any bias for regions that are more easily sonicated. You can at least run ChIP and see how much DNA you're able to get using pico-green or something similar, and if needed, you can pool more than one ChIP of the same type to get more DNA.
Edited by KPDE, 30 April 2012 - 07:33 AM.
Posted 06 May 2012 - 09:47 PM
20x20s bursts, and 30x20s bursts
Very happy with how it has sheared this time
Posted 07 May 2012 - 12:26 AM
Posted 09 May 2012 - 10:54 AM
Posted 09 May 2012 - 03:14 PM
Use this Chip Protocol
The above protocol is a bit little changed of
Farnham Lab MicroChIP Protocol
Posted 13 May 2012 - 08:24 PM
The lysis buffers in that document are already the same ones that I am using, memari.
Posted 24 May 2012 - 05:07 PM
And you should use a nuclear fraction to do it.
Use Cytoplasmic Lysis buffer to break the cell and then use nuclear fraction with a nuclear lysis buffer that contain 1% SDS.