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Shearing (sonication) problem for ChIP - Using Bioruptor (Jun/10/2009 )

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thanks for the input! But it is really hard just to shear the cells! I am still working on it.

But someone in the lab did 40 minutes (40 cycles) of sonication using Bioruptor and I can see a strong band at around 200 to 500 bp. No smearing at all! It is clean shearing for me. Only 200 to 500 bp. But I do think 40 minutes are abit too long? Especially one is using Bioruptor? We always do 10 minutes interval and change the ice. I wonder if that makes the shearing efficiency to drop?

Thanks!

-timjim-

timjim on Jun 23 2009, 04:21 AM said:

thanks for the input! But it is really hard just to shear the cells! I am still working on it.

But someone in the lab did 40 minutes (40 cycles) of sonication using Bioruptor and I can see a strong band at around 200 to 500 bp. No smearing at all! It is clean shearing for me. Only 200 to 500 bp. But I do think 40 minutes are abit too long? Especially one is using Bioruptor? We always do 10 minutes interval and change the ice. I wonder if that makes the shearing efficiency to drop?

Thanks!


If someone did 40 minutes without changing the ice, I'm guessing the ice was melted by the end. What probably allowed the shearing to work was that all the ice melted. The ice severely reduces sonication efficiency for us. In fact we found it impossible to get good shearing with ice in the bath. You might try sonication with short intervals, replacing the water with ice cold water each time. As I mentioned above, we developed a simple water cooling system and that solved all our shearing problems.

-KPDE-

well, we change the ice every 10 minutes (so in total it is 40 mins), sometimes we get sheared band sometimes we dont. And they are the same samples!!! I really dont understand the cells sometimes. Getting irritated by the stupid cells. =p

I dont really understand the cooling system, so basically, one should just pump the water out and replace with cold water? I would like to make such cooling system too. Well, you can even buy it from Diagenode....

-timjim-

Hi Timjim,

The issue with using a sonicator for chromatin shearing is with reproducibility of settings with different cell lines. All shearing conditions have to be optimized for each cell line which can be quite a laborious task in itself. Thermal damage is a major issue when dealing with smaller shearing sizes. Thermal denaturation of the the proteins, non-specific and biased shearing, thermal acetlyation issues are commonly noticed.
Have you heard of the Covaris AFA technology for non-contact, isothermal chromatin shearing? Here are some protocols using the Covaris technology http://covarisinc.com/dna-shearing.html. Let me know if you need further details. Good luck.

hamid

-hamidk-

hamidk on Jul 12 2009, 05:42 PM said:

Hi Timjim,

The issue with using a sonicator for chromatin shearing is with reproducibility of settings with different cell lines. All shearing conditions have to be optimized for each cell line which can be quite a laborious task in itself. Thermal damage is a major issue when dealing with smaller shearing sizes. Thermal denaturation of the the proteins, non-specific and biased shearing, thermal acetlyation issues are commonly noticed.
Have you heard of the Covaris AFA technology for non-contact, isothermal chromatin shearing? Here are some protocols using the Covaris technology http://covarisinc.com/dna-shearing.html. Let me know if you need further details. Good luck.

hamid

Hello to you all


I have a dumb question, in terms of sonication efficiency what is the difference between 30 sec on/30 sec off cycles and smaller cycles, like 15 sec on/15 sec off?

Thanks

-Radish-

Radish on Feb 5 2010, 09:30 AM said:

hamidk on Jul 12 2009, 05:42 PM said:

Hi Timjim,

The issue with using a sonicator for chromatin shearing is with reproducibility of settings with different cell lines. All shearing conditions have to be optimized for each cell line which can be quite a laborious task in itself. Thermal damage is a major issue when dealing with smaller shearing sizes. Thermal denaturation of the the proteins, non-specific and biased shearing, thermal acetlyation issues are commonly noticed.
Have you heard of the Covaris AFA technology for non-contact, isothermal chromatin shearing? Here are some protocols using the Covaris technology http://covarisinc.com/dna-shearing.html. Let me know if you need further details. Good luck.

hamid

Hello to you all


I have a dumb question, in terms of sonication efficiency what is the difference between 30 sec on/30 sec off cycles and smaller cycles, like 15 sec on/15 sec off?

Thanks


Back when I had access to a Misonix 3000 I remember watching the power output reading during sonication and noticed that power decreased from the beginning to the end of a pulse. It seemed the longer the pulse the lower the average power output (since there was more time for the power to decrease). I used 5 second pulses after that since the power output stayed around 100% of what I set it to. I don't know if this is the case with all sonicators though.

-KPDE-

KPDE on Feb 5 2010, 04:21 PM said:

Radish on Feb 5 2010, 09:30 AM said:

hamidk on Jul 12 2009, 05:42 PM said:

Hi Timjim,

The issue with using a sonicator for chromatin shearing is with reproducibility of settings with different cell lines. All shearing conditions have to be optimized for each cell line which can be quite a laborious task in itself. Thermal damage is a major issue when dealing with smaller shearing sizes. Thermal denaturation of the the proteins, non-specific and biased shearing, thermal acetlyation issues are commonly noticed.
Have you heard of the Covaris AFA technology for non-contact, isothermal chromatin shearing? Here are some protocols using the Covaris technology http://covarisinc.com/dna-shearing.html. Let me know if you need further details. Good luck.

hamid

Hello to you all


I have a dumb question, in terms of sonication efficiency what is the difference between 30 sec on/30 sec off cycles and smaller cycles, like 15 sec on/15 sec off?

Thanks


Back when I had access to a Misonix 3000 I remember watching the power output reading during sonication and noticed that power decreased from the beginning to the end of a pulse. It seemed the longer the pulse the lower the average power output (since there was more time for the power to decrease). I used 5 second pulses after that since the power output stayed around 100% of what I set it to. I don't know if this is the case with all sonicators though.


WOW, nice one, I am going to start sonication in a few minutes I will pay attention to it.
I read somewhere that shorter pulses were more effective, I was thinking about trying this to.

thanks KPDE!

-Radish-

Hi everyone,

I have a slightly thick question...I am using the misonix 3000 cuphorn to sonicate crosslinked cells for ChIP. How far away does the tube have to be from the metal platform? like 1cm, 0.5cm or 2mm? I am having difficulty in getting all my DNA fragments in 200-600bp range. I have DNA in this size range, but I also have a large 3000bp fragments as well...need to get rid of this. Just wondering the distance between the tube and the platform affects this?

Thanks so much in advance!

-Hedgehog-

Hy everyvody,

I have a problem that i don´t undestand, I did the quantitative PCR with sybr green for measuring my DNA after ChIP, and i get signal, when i look for the melting curve for the amplifications it seems to be ok,

But i wanted to comfirm the amplicones are the size i was searching for and i have run an agarose gel with my samples.

I have had no signal! how it is possible? the amplification in the quantitative PCR was really good, the CTs of the samples was between 24 and 30....

May be possible that sybr green union to the DNA makes it impossilbe to get the ethidium bromure and get the fluorescence?, it´s one week since i did the PCR but the plate has been in the freezer anytime ..

Anyone can explain me? now i am doubty of my results...... i must have seen the bands, didn´t I ?

Thanks.

-Fe Li-

Recommendation for CHIP:
1-Chip lysis buffers usually have 1% SDS. The breaking of chromatin by lysis buffers that have no SDS is very hard. In order to check, you can use this buffer from this paper:
http://www.nature.co...ot.2006.27.html
It is the most awful buffer that I have ever tested. Sonicating yourself is easier than breaking chromatin by that lysis buffer.

You can decrease SDS percentage up to its Critical micelle concentration (CMC)
http://openwetware.o...entration_(CMC)

2-Decrease the power of sonicator to between 30 to 50.

3- Volume is important. The less volume, the better sonication. Do not sonicate more than 500ul in vial.
4-Bioraptor needs transparent vials(TPX tubes).

-----
Babak Memari

-memari-
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