sonication - no DNA shearing (Dec/05/2008 )
Hi
I am doing my first ChIP and trying to sonicate the DNA with a bath sonicator.
But the DNA just stays intact.
Yesterday I diluted the sample (more nuclear lysis buffer) and sonicated up to 30 time (on 30" /off 30").
And I can see only mild shearing, majority of the DNA remains intact.
I dont find anybody discussing this type of problem... is there nobody with same problem;
what could protect the DNA from shearing??
krhim
-krhim-
QUOTE (krhim @ Dec 5 2008, 06:21 AM)
Hi
I am doing my first ChIP and trying to sonicate the DNA with a bath sonicator.
But the DNA just stays intact.
Yesterday I diluted the sample (more nuclear lysis buffer) and sonicated up to 30 time (on 30" /off 30").
And I can see only mild shearing, majority of the DNA remains intact.
I dont find anybody discussing this type of problem... is there nobody with same problem;
what could protect the DNA from shearing??
krhim
I am doing my first ChIP and trying to sonicate the DNA with a bath sonicator.
But the DNA just stays intact.
Yesterday I diluted the sample (more nuclear lysis buffer) and sonicated up to 30 time (on 30" /off 30").
And I can see only mild shearing, majority of the DNA remains intact.
I dont find anybody discussing this type of problem... is there nobody with same problem;
what could protect the DNA from shearing??
krhim
I have not done ChIP assays myself, but I would ask yourself what the purpose is of the sonication step. Is it to break up the DNA into small fragments? If so, can you do this enzymatically? Can you sonicate it with a microprobe rather than a bath sonicator? Can you sonicate for longer or increase the frequency?
If something is not working, I always ask what the purpose is for the step, as there are often many other ways to acheive the same goal. Hope that helps!
-Cheamps-
QUOTE (krhim @ Dec 5 2008, 03:21 AM)
Hi
I am doing my first ChIP and trying to sonicate the DNA with a bath sonicator.
But the DNA just stays intact.
Yesterday I diluted the sample (more nuclear lysis buffer) and sonicated up to 30 time (on 30" /off 30").
And I can see only mild shearing, majority of the DNA remains intact.
I dont find anybody discussing this type of problem... is there nobody with same problem;
what could protect the DNA from shearing??
krhim
I am doing my first ChIP and trying to sonicate the DNA with a bath sonicator.
But the DNA just stays intact.
Yesterday I diluted the sample (more nuclear lysis buffer) and sonicated up to 30 time (on 30" /off 30").
And I can see only mild shearing, majority of the DNA remains intact.
I dont find anybody discussing this type of problem... is there nobody with same problem;
what could protect the DNA from shearing??
krhim
What kind/brand/model of sonicator are you using?
-KPDE-