DNA sonication - RNA contamination? - New probe, start all over (Mar/17/2010 )
epi123 on Mar 26 2010, 05:51 AM said:
Good to know, but i will be using the magnetic DNA purification beads after my IPs.
hi!
can any body explain how sonication parameters(amplitude,pulse on,pulse off) work..I am keen on knowing the interrelation between all three?
can anybody refer to any experiment keeping one constant and vary others?
we're trying to sonicate DNA for ChIP using Ez-ChIP's protocol(not the kit)
sameer
sameerbau on Sun Nov 7 18:47:57 2010 said:
hi!
can any body explain how sonication parameters(amplitude,pulse on,pulse off) work..I am keen on knowing the interrelation between all three?
can anybody refer to any experiment keeping one constant and vary others?
we're trying to sonicate DNA for ChIP using Ez-ChIP's protocol(not the kit)
sameer
2 more questions..
1.is 25% amplitude equal to 25% power.our sonicator(i forgot the name) has fixed power at 150watts.
2.for ChIP,while standaridizing crosslinking,is it necessary to decrosslink? what we were trying was simply scraping-sds lysis-phenol chloroform-ethanol precipitation-agarose gel.
and the last..one more..
is it necessary/recommended(while:
1.sandardizing sonication conditions,
2.going for the full on chip protocol)
to centrifuge after you sonicate to get your sheared chromatin?
i ask because in some of the samples,there's a white precipitate after ethanol precipitation and centrifuging at4 degs.
and can somebody provide a complete guide to probe based sonication of eukaryotic genomic DNA?
thanks a lot!!