Doubts on Sonication in the ChIP - Not enough sonication? (Sep/06/2005 )
I have been sonicating chromatin without foaming problems, using 10 continuous cycles of 20'' at a setting of 80% (using a UP100H sonicator). After controling the sonicated chromatin on a 1% agarose gel, there is an smear but most of the sample gets stucked in the well. My question is if this means I need to sonicate more? Maybe considering the chromatin is crosslinked with proteins is normal that gets stucked there? I am affraid to affect the proteins by sonicating more. Someone can give me a hint?
sounds like you need more sonication to me.... did you freeze your chromatin before sonicating??
Thanks for answer.
No I have not frozen the chromatin before sonicating...
I can try to sonicate more, because really the most of the chromatin is in the well.
I send to you attached the image...
Did you do a reverse crosslinking and phenol extraction of the DNA after sonication before running the gel? It sounds like the DNA was not purified. The condition you gave should be able to break DNA into desired pieces.