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