ChIP: Sonication - (Dec/16/2005 )
I have been using EZ ChIPTM protocol from Upstate. I have been trying to find the optimal conditions for sonication, but I have problems to do it. I use an ultrasonic homogenizer, model 150 V/T (Biologics) at about 40% of maximum power (I suppose it, because the LED doesn’t work) and at 50% pulser. I sonicated from 70 seconds until 15 seconds, but when I run a gel, there is too much DNA that isn’t sonicated.
hi are you getting any foaming in your protocol? if so this is probably why.
shearing efficency reduces with foaming, check out the pinned topic
When I got foaming I saw that DNA wasn’t sheared. But, I’m not usually getting foaming, and I saw a smear, but most of the DNA isn’t sheared enough.
I think the problem is that the sonication time is not enough. You are using modest power setting as indicated by no foaming. I would suggest that you increase the total time to 150 second, that is a series of 15x10" bursts, pause for 10"-20" between every burst.
After sonication you have to do a reverse crosslinking and purification before running the gel.
Sorry, but I had a mistake in the first message. I sonicated until even 13 minutes and I still obtained fragments higher than 1000 bp. And after socincation I did a reverse crosslinking and I added RNase and Proteinase.
And, on the other hand, is the sonication dependent on cell type? Because, I work with cells in suspension (Jurkat T cells). Could it be the reason of my problems?
Thanks for your replies, I'll try to check it another time.
In what volume you do the sonication? Both volume and cell type can affect sonication efficiency.
I do the sonication with a final volume between 300-500 microliters.
Could it be that with my cells I have over-crosslink? I use a final concentration of 1% of formaldehyde. Maybe I must try with lower concentrations, musn't I?
or shorter times, instead of 10 minutes, 5 minutes. over fixation will affect IP efficiency.