ChIP sonification woes - why is my DNA cut into tiny pieces?? :( (Oct/06/2008 )
Hello to all you Chippers (lame joke) out there!
So im working with MEFs and up to now no one can figure out why the shearing step doesnt work, we use the Bioruptor from diagenode, have tried 7,5 10 20 min with different volumes but still we get an enrichment of very small fragments (100-200 bp). We also used 0.5% formaldehyde and 1 % formaldehyde and two different pulses (10 and 20).
Ive done a ChIP before with a branson sonicator and i only needed 2 min with 5 second pulses. I dont understand, maybe someone else out there knows the Bioruptor and cant tell me what the deal is? Less cells in larger volume? Less time with shorter pulses? We really need this thing to work in order to show that our protein binds invivo to a certain target 
If only I could make this happen, since id done ti before it was my idea to use different formaldehyde concentrations and volume and shorter pulses but it still didnt work. I think it just made me look like a moron yet again
Do you mean your fragments are too small or too large?
If they are too small, then sonicate for less time. 10 and 20 minutes may be overkill, 7.5 may be too.
I get small fragments, like 100 or 200 bases long and a slight smear going all the way up the gel. Would doing 5 min with 5 seconds on 5 seconds off be an idea??
Its just a bit strange, they been trying tog et it right here for a while now, and they always used a long time with 20 second on 20 second off pulses. When I suggested less time i was looked at funny, and when my 10 min idea didnt work it was kind like yeah i told u so.
But neways I agree it might be overkill, I just thought maybe the bioruptor needed more time for some reason
I agree,
sonicate for less 
What cell conc are you using? We typicially sonicate for 5 minutes at 1 million cells/30ul.
Clare
Uhm, I had about 2 million cells in 400 microliters, weve also used about a million cells in 100 microliters.
What does the small volume do?? Do u use a Bioruptor too?
What does the small volume do?? Do u use a Bioruptor too?
Try increasing the concentration of your cells. That should work. Yes, we are using the Bioruptor and typically sonicate in a total vol of 1.3ml (in 10ml tubes) with a cell conc of 1 million cells/30ul.
Hope that helps
Clare
I used a standard Branson-250 sonifier, approx 10 million cells in 400ul, 30 seconds on, 30 sec off for 8 mins, with a duty cycle at 40% from memory.
So, 8 x 0.5 x 0.4 = 1.6 minutes of total sonication over 8 minutes.
All cells are different but I hope this is useful for you.
Interesting........when I did a ChIP before we were going to do ChIP-chip so we needed a lot of material, we also used about 10 to 50 milion cells in either 500 or 1000ul. Maybe I really should try to increase concentration..........
thank u!!!!! I hope it works, or at least I see a slight improvement that could point me in the right direction !