17 replies to this topic

[KPDE]

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.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.

[Radish]

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.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!

[Hedgehog]

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!