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Shearing issue: always having unsheared DNA (Please help!) - (Apr/26/2012 )

Hi ChIP experts;

It's now two months that I've been stuck in the shearing part of the ChIP assay. I'm using cup horn settings but I always have lots unsheared DNA and a perfect smear around 400bp. I have gone from 1 min of sonication to over 40 min but I still do have that unsheared part. I have tried anything that the company suggested: polystyrene tubes, decreasing the sample volume, distance from the horn, etc. However, it is still a big mess. I'm really exhausted and don't know what to do. I would be very very very thankful if any of you have any suggestion.
Ps. I'm doing ChIP on MCF-7 cells

Best
Amir

Attached Image

-a_bahreini-

Before you reverse the crosslinks and ProK treat your samples spin your sheared chromatin down @ 14,000xg for 10min and take the supernatant. Use this supernatnat to determine your shearing efficency.

-chabraha-

Thanks for your response. Normally, I don't treat my samples with ProK before purifying them. Actually, I use Qiagen PCR Purification kit and I don't think I need to treat my samples with ProK. I spin my samples after sonication but I always get this unsheared part :(

-a_bahreini-

Do you know if your reverse crosslinking has gone to completion? Maybe try the ProK step just for comparison

-chabraha-

This is how I reverse cross-link to check shearing:
I use 10 ul of my sonicated sample and add 40 ul IP elution buffer (50 mM NaHCO3 & SDS 1%).
Then I add 2 ul of 5 M NaCl
I boil them for 15 min
then I add 5 ul of 3M NaOAc ph=5
and then I purify them with PCR kit

-a_bahreini-

i had a similar type of smear when I checked my crosslinking via the Chelex method............even after boiling for more than 20min. I switched to a different method (a bit longer, ~3hrs, but better in my opinion)............I was doing my ChIPs on mouse embryo fibroblasts.

-chabraha-

I don't know but I will try that. Did you boil your samples for 3 hours or put them at 65 for 3hrs?

-a_bahreini-

Instead of the Chelex.................I resuspended my sample in a Tris-SDS buffer containing B-mercaptoethanol. Heated it at 99 degrees for 1hr. Cooled the sample, and added 20ug ProK and incubated the sample for 57 degrees for 1hr and then Phenol/Chloroform extracted the DNA and precipitated it with EtOH.................I'm pretty sure I've posted the exact method somewhere in this forum.

-chabraha-

why do you use polystyrene tubes?
should not you use polyPropylene tubes(Non_Sticky)?

Babak

-memari-

Those polystyrene tubes are the best for the transmission of energy in cup-horn method

-a_bahreini-