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Sonication Problems, Branson 450, LNCaP cells


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#1 LexLuthor

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Posted 17 April 2009 - 02:00 PM

Haven't been able to find an answer to this problem, so hopefully someone can help...

I am currently doing ChIP assays using LNCaP cells using the following conditions:

-after lysing the cytoplasmic membrane etc, ~3.0x10^7 cells sonicated in 500 ul of lysis buffer (140mM NaCl, 50mM HEPES pH 8.0, 1mM EDTA, 1% Triton, 0.1% SDS, 0.5% Na Deoxycholate)
-samples are sonicated using a Branson Sonifier 450 (Duty Cycle 90%, Output 4)

Here's the problem: when cells are fixed with 1% HCOH (10min at rm temp. in media followed by quenching with glycine) I cannot completely shear all of the large fragments of chromatin--there's always a smear of DNA all the way up to 10-12 Kb when run on an agarose gel. When sonicating, I usually do cycles of 8 pulses (or seconds) with cooling on ice between cycles. I've tried from 8x8 up to 30x8 with no reduction in the average size. I tried increasing the Output to 6 and sonicating 8x8 up to 18x8 but with no luck in reducing the average size of the DNA. I've tried tried fixing in different concentrations of HCOH (0.2, 0.4. 0.6. & 0.8%); at 0.2 and 0.4% I have no problem sonicating ALL of the chromatin down to a size between 200-800 bp using Duty Cycle 90%, Output 4 and 12 cycles of 8 pulses. But, at the two higher concentrations I can't get the same results--there's always bigger fragments that remain.

Does anyone have any suggestions? It seems wrong that I would have to fix in a lower % of HCOH--I've never seen anyone report fixing in a concentration lower than 1%. Is sonicating in 0.1% SDS is really that inefficient that I can't reduce the average size down below 1000 bp? Or is it some other condition affecting sonication efficiency?

Any suggestions would be greatly appreciated, especially if you're familiar with the Branson machine.

Thanks!

#2 pcrman

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Posted 17 April 2009 - 07:03 PM

To me your output of 4 is kind of high. Did you get foaming? If yes, because foaming affects sonication efficiency you can reduce the output to 2. What kind of tubes did you use for the sonication?

#3 LexLuthor

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Posted 18 April 2009 - 08:08 AM

To me your output of 4 is kind of high. Did you get foaming? If yes, because foaming affects sonication efficiency you can reduce the output to 2. What kind of tubes did you use for the sonication?



Never had any problems with foaming and I always use 1.5ml microcentrofuge tubes.

#4 JPchip

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Posted 29 April 2009 - 04:22 PM

I've got the same sonicator and am also a little surprised you don't get foaming. If I use output 4 I don't just get foaming, I get a shower in whatever cells were in the tube.

We do sonicate in very different buffers though. I use SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1). Have you checked your nuclei are lysed in only 0.1% SDS under a microscope? I have a vague recollection of using 0.1% SDS and finding it didn't lyse nuclei as well as it should. If most of your sonicator power is spent on breaking open nuclei this could be part of the problem.
More likely though, I think you're trying to sonicate too many cells in one tube. 1x10^6 is perfectly sufficient for ChIP, and in truth I've successfully ChIP'ed with far less. Such a large amount of chromatin contained in so many cells could could also explain why your sonicator is having trouble.

If all else fails you could always try fragmentation with micrococcal nuclease (MNase) instead.

#5 LexLuthor

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Posted 04 May 2009 - 02:00 PM

Thanks so much for your reply JP.

It's interesting that your results are so different from mine when using the sonicator at Output 4. We routinely sonicate our whole-cell protein lysates, which are lysed in 200-400 ul of 2X sample buffer (2% SDS), using an Output between 4-5 and we never have a problem with foaming etc. It makes me wonder if there is a problem with our sonicator. Are you using a double-step micro tip?

As for the buffer itself, you are correct that it doesn't work very well at lysing the nuclei of the fixed cells, although I tested it on unfixed cells and it works fine. I have been concerned about that, although even after 'extended' sonication I don't get much change in the average size of my chromatin. So I'm not sure how other people are able to successfully use this buffer.

Do you use 1x10^6 cells/IP or is that your total number of cells you lyse and soncicate? And what volume do you sonicate in? Typically, I use ~3x10^6/IP so I get about 10 IPs per plate of cells.




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