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
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.
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
Do you know if your reverse crosslinking has gone to completion? Maybe try the ProK step just for comparison
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
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.
I don't know but I will try that. Did you boil your samples for 3 hours or put them at 65 for 3hrs?
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.
why do you use polystyrene tubes?
should not you use polyPropylene tubes(Non_Sticky)?
Those polystyrene tubes are the best for the transmission of energy in cup-horn method