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Shearing (sonication) problem for ChIP

sonication shearing chip

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28 replies to this topic

#16 KPDE

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Posted 05 February 2010 - 03:21 PM

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.co...a-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.

#17 Radish

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Posted 05 February 2010 - 04:38 PM

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.co...a-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!

#18 Hedgehog

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Posted 16 April 2012 - 04:39 AM

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!

#19 Fe Li

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Posted 18 December 2012 - 04:39 AM

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.

#20 memari

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Posted 18 December 2012 - 08:10 PM

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

Edited by memari, 18 December 2012 - 08:20 PM.

-----
Babak Memari

#21 chabraha

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Posted 20 December 2012 - 08:53 AM

you should be able to confirm the size via the Tm..........often times primer design programs will give you a theoretical Tm for the PCR product...........you can also calculate this yourself if you do not have a program..........but I think IDT's website can do this for you.

With respect to your question........the possibility exists that you did not expose the gel long enough to see the small products..........and the possibility you bring up is also good...........use a syber green appropriate filter on a chemidoc imager and you'll probably see your products.
Treasure Chest Wizardry

#22 maglie

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Posted 17 July 2013 - 06:30 AM

Dear all,
i have big problem for preparing a good chromatin for chip analysis.
do you have a good protocol for sonicate activated naive cd4 T cells?
i have in my lab Bioruptor sonicator....i check many lysis buffer and many protocols but i'm not able to have genomic fragment ranging from 500nt to 100nt.
PS: i think my problem is no the lysis (cells are nicely lysated as observed by microscope with trypan blu!!!) but the sonication condition.

thanxxxxx

#23 uhsiracu

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Posted 17 July 2013 - 07:10 AM

I don't use bioruptor, but I usually sonicate my CD4+ lymphocytes at 30% power for 5 cycles, each one of 10seconds. Between each cycle I leave the sample on ice for 30-40sec. In which volume do you sonicate?

#24 maglie

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Posted 17 July 2013 - 09:21 AM

200 microl

#25 maglie

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Posted 17 July 2013 - 09:22 AM

are u using RIPA buffer for lysis/sonication?

#26 uhsiracu

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Posted 17 July 2013 - 09:37 AM

200ul should be fine, I also do that way. No, I'm using 50ul SDS lysis buffer for lysis (it contains 1% SDS) and then I add 150ul of Chip dilution buffer that contains 0.1% SDS: I obtain fragments around 300-500bp.

#27 maglie

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Posted 18 July 2013 - 06:05 AM

thanx!

#28 uhsiracu

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Posted 18 July 2013 - 07:53 AM

I'm sorry, I missed a 0! The chip dilution buffer contains 0.01% SDS. Anyway, you are welcome!

#29 rajanagre

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Posted 01 August 2013 - 08:20 PM

I am standardizing the sonication condition for tissue ChIP assay, I would like to know the fragment (smear +bright band) is from DNA or from RNA contamination, I did RNase treatment; the lane after ladder is the sample without reversing the cross linking. please suggest me.

 

Raja

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