9 replies to this topic

[varma_ran]

hi everyone
i am having a problem with chip assays following sonication with bioruptor.
i have done the same chip assays successfully before but i was sonicating with probe sonicator.
Since now i have to prepare the samples for chipseq,  we have got bioruptor 400 in our lab along with water bath to mintain temp at 4 degrees.
i am using 15 cycles (30sec on 30 sec off) with bioruptor and exactly same fixation and chip protocol as i have used before with mouse cortex and pol2 antibodies. I am using millipore ez magna chip kit (cat no-17-409).
So the only difference now is sonicating with bioruptor (fragments of 100-600bp)instead of probe sonicator and my chipqpcr does not work even with the positive control primers (b-tubulin, pcr product size 100bp).
please help me solve this issue.
Thanks very much...
i have enclosed my chip-protocol

Attached Files

•  G-CHIP-PROTOCOL.doc   30K   194 downloads

[Mighty Mouse]

Hi Varma,

So I've used Millipore's kit with the BioRuptor and had it working (I eventually moved to making the reagents myself to save a few bucks, but the protocol was very similar to what Millipore put together). The big difference was that I only ran 5 cycles, not 15. So I had it set on high and ran it for 5 min of 30s on and 30s off. Your issue might be in part that your samples are getting too hot. When I ran the experiments the BioRuptor was placed in a cold room, nonetheless after each five minute bout with my samples I put ice in the water bath and let it sit for 10 or so minutes to cool down again (then removed whatever ice was left so as not to decrease my sonication efficiency from set to set). I imagine after 15 min of 30s on 30s off the water bath would be quite hot. Whenever I was interested in doing more cycles (e.g, when I was playing around with higher concentrations of formaldehyde) I would run for 15 min of 30s on and 1 min off to allow the water to cool down just a little bit more.

Looking at your protocol I'm noticing one of the other differences between your protocol and mine (I was also working in mouse brain, hippocampus actually) is that I homogenized my tissue and removed the cytosolic fraction prior to sonication. Doing something like this might increase your sonication efficiency by removing a bunch of unnecessary cellular debris thereby allowing you to decrease your sonication time and not heat up your samples as much.

There is also one other potential issue that you might want to consider. I used Millipore's magnetic beads for all of my ChIP and I have found A LOT of variability from lot to lot with their beads (in retrospect I probably would have used another company like ActivMotif's beads perhaps). Recently we had some ChIPs not work out and I suspect it was due to crappy beads as I had run through the ChIP many times with the same lot of antibody and had it working with a different lot of beads. If you have some beads from a previous lot hanging around that you know have worked it might be worth examining.  


I hope some of these things help, certainly let us know!

Regards,

MM

[varma_ran]

Hi MM
thanks very much for your suggestions, i am going to try these soon and will let you know.
I have some more queries, could you please provide for your suggestions.
Our bioruptor is attached to waterbath that has water maintained at 4 degrees and there is continuous circulation of water from this water bath to sonication bath so that the temperature of water inside sonication bath is also maintained at 4 degrees, do you still think that the heating of samples could happen?
Do you think having smaller fragment sizes (100bp-200bp) will not bind that sufficiently to beads (from millipore kit) as compared to longer fragments and thus there is loss of chromatin during this step and further during final purification.
could you please let me know the fragments size with bioruptor that worked fine with bead from millipore kit for you.
Also per one chip reaction, what is you your chromatin to antibody ratio.
Thanks again
VR

[jonny_boy]

Hey Varma,

I also have a few suggestions based on my experience with the biorupter. First of all, have you run out the products resulting from your sonications on a gel to check shearing? As mighty mouse suggested you may well have gone too far with your sonications by 15 cycles. Generally when working with a new cell line I first optimise my sonications by removing a small aliquot after each cycle and running out these aliquots on an agarose gel after reverse crosslinking. In this way I usually see no improvement in the level of fragmentation after 8 cycles using the biorupter.

Secondly, have you tried doing a PCR at another point in your region of interest? There is a chance that changing your sonication conditions could have altered the resolution of your assay, the signal you observed previously could have been due to pull down at a region distal to your PCR product but still included on the larger fragments produced by probe sonication.

Just a few ideas!

Jon

[jonny_boy]

Oh and one more thing! The size of the DNA fragments would not affect binding to the magnetic beads as these are actually binding to antibody attached to protein - fragment size should not really affect this reaction.

Jon

[varma_ran]

Hi Jon
thanks so much for your suggestions, i am definitely going to try for less number of cycles with bioruptor so as to not get too small fragments and also avoid the heating problem if any.
VR

[varma_ran]

Hi Jon
sorry i forgot to answer, yes i checked my fragments after sonication, it varies from 100-600bp (chromatin without reverse crosslink)and the pcr primers i am using is for b-tubulin (positive control, pcr product size 100bp) and antibody used is RNA pol2.
I think having too small fragments might have some problem, i am going to try less number of cycles so that fragment size is not below 200bp.
please let me know if you have any more suggestions.
Thanks
RV

[Mighty Mouse]

varma_ran, on 08 December 2010 - 05:32 AM, said:

Hi MM
thanks very much for your suggestions, i am going to try these soon and will let you know.
I have some more queries, could you please provide for your suggestions.
Our bioruptor is attached to waterbath that has water maintained at 4 degrees and there is continuous circulation of water from this water bath to sonication bath so that the temperature of water inside sonication bath is also maintained at 4 degrees, do you still think that the heating of samples could happen?
Do you think having smaller fragment sizes (100bp-200bp) will not bind that sufficiently to beads (from millipore kit) as compared to longer fragments and thus there is loss of chromatin during this step and further during final purification.
could you please let me know the fragments size with bioruptor that worked fine with bead from millipore kit for you.
Also per one chip reaction, what is you your chromatin to antibody ratio.
Thanks again
VR

Hi VR,

Sure thing...so if you have cold water circulating you are probably fine with regards to temperature, sounds like a good setup to have for this sort of thing. For the fragment sizes, I typically had a good smear between 200 - 1000 bps. I don't know that it's necessary to get such small fragment sizes as you have; if you want to really ramp up your resolution you can also consider doing an enzymatic digestion; would probably be safer with respect to preserving your factor-DNA interactions.

For my ChIP reactions I used 2ug of DNA (so at the same time as pulling an aliquot for checking shearing efficiency I would also clean up the prep to purify the DNA and measured its concentration). The amount of antibody I used depended a lot on the specific antibody. If the manufacturer had a suggestion I usually just used that and maybe doubled it if I was having some difficulty. If the manufacturer has not tested the antibody in ChIP I would usually use somewhere between 2-10 ug of antibody in a 500 uL volume, trying a couple of different amounts; it's imperative for this sort of testing of unproven antibodies to have good negative controls like a negative control region and IgG.

Hope this helps.

MM

[varma_ran]

Hi MM
thanks very much for your suggestions. i am currently trying the same to avoid very small fragments, that might be one of the issues responsible for the failure.
VR

[hamidk]

Hi,

You should always run a western on a portion of your sheared chromatin to make sure you have not destroyed your epitope in the shearing process. That way you don't waste time and reagents on IP, qPCR, and sequencing.

Thank you

hamid