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ChIP assay - Foaming in sonication and other woes - (Jan/27/2005 )

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QUOTE (sridhar @ Jan 10 2006, 09:16 AM)
Dear Pcrman,

Is a water bath sonicator good for shearing DNA in a CHIP assay??. I use "SUPER RK 103H from Schütt labortechnik, i sonicate DNA at 100 strenght for 2 mins + 2 mins in ice cold water (i place the samples on ice after the first 2 mins). The DNA smear on the gel looks the same with sonication strenghts of 1.5+1.5 mins or 2+2 mins or 3+3mins. Could you please explain.

Thank you.

Hi Sridhar,

I have never used a water bath sonicator, but I think no matter what sonicator you use, the goal is to shear DNA into 200-1000bp fragments while minimizing sample heating during sonication. If the goal can be reached, anything will do.


QUOTE (xiongbaobao @ Jan 3 2006, 11:44 AM)
hi everybody, I just started to do the CHIP assay, I also have problems about shearing the chromatin DNA. so I join this forum for help

the following is my procedure.
1: I isolated the hippocampus of rat then minced in a 35mm plate filled 4 ml cold PBS
2: then I add the Formadehyde to a final concentraton of 1%
3: I incubate the plate at 37 C for 15 minutes(because the PBS is cold, so prolonged the incubation time)
4: I isoated the nuclear then lysis it in SDS lysis buffer.
5: and then I sonicate the lysis, the total volume I used is 800 ul.

I tried many sonicte conditions, the power setting range from 20% to 30% (KS 600 sonictor) the total time range from 60sec to 180sec (usually I will sonicate 20sec or 15sec, and then brake for 2min), I must note that there is no foaming when I did it.
but the sheared DNA is not good. there is a bright band at 4KD.

in addition, I found that the bad shearing will not enfluence the CHIP relults, I mean I can also get a positive band after PCR about 30 cycles (but not in the no antibody control), why this happened?

hope for your help blink.gif

The volume has a big impact on sonication efficiency. 800 ul seems too much to me. Try to reduce the volume to 400 or 200 ul.

Yes, you can still get positive band even sonication is not enough because your antibody is pulling down a big piece of DNA associated with the protein. The positive signal may not necessarily mean that the DNA area of your interest is associated with the protein.


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