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Sonication trouble


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#1 rockpaperscissor

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Posted 07 December 2009 - 02:36 PM

Hi,

This is my very first post :lol:. I am working on Mouse Granulosa cells and we have been planning to do ChIP-CHIP. Our lab protocol combines both Mnase and sonication to get 200-1000bp range. Mnase treatment is done at 300 U on 12 million cells, 1ml volume for 10/15 minutes and the reaction is stopped using EDTA. I then transfer 4 million cells and make up the volume to 700 microliter and using Vibra-Cell™ ( http://www.sonics.bi...t/VC505-750.pdf ) I sonicate at a 10 second pulse with an interval of 30 seconds for 10, 12 or 15 times on ice. Unfortunately I have not been able to get my DNA to stick between 200-1000 bps. I keep getting larger amount of mononucleosomes. I have tried reducing the number of cells to 1 million and decreasing the Mnase concentration from 300 U to 15 U but I keep hitting the wall. Either it gets digested too much or very less :D . I am stuck at these extremes. The biggest problem is the availability of cells, as I get an average of 15 million cells per 12 animals. I have attached the image of my gel. I have been thinking i should stop Mnase completely and just try sonication. I have already spent more than 2 months on this, any thoughts or insights will be highly appreciated. Thanks.

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#2 gogreen

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Posted 08 December 2009 - 06:06 AM

hi there,

I was using the sonics Vibracell for ChIP from Human cell lines without any MNase digestion...I remember I used to sonicate it for 30 cycles of 20-30 sec ON and 40 seconds-1min OFF and get a distribution of which over 70% was 200-1kb...That should still be good enough fo ChIP as 100% efficiency of sonication is just a dream!! . From your gel, it looks like the MN already cuts most of the chromatin in range of 10-200 bp..did u ever try only sonication and only MNase to see the difference.

Is the gel pic provided here after the decrosslinking? If not, I would suggest you to decrosslink it and run a gel 'coz its quite deceptive seen on gel without decrosslinking. The fragments turned out to be smaller after decrosslinking and a cleanup in my case..maybe because of all other muck in the sample hindering the movement!

Edited by gogreen, 08 December 2009 - 06:07 AM.


#3 rockpaperscissor

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Posted 08 December 2009 - 09:14 AM

Thanks for the reply Gogreen. I have not tried just sonication with these mouse cells. I have tried Mnase treatment from 7-30 minutes, but either it gets all digested to mononucleosome or they have large chunks at higher bps. The gel image you see is after crosslinking and phenol-chloroform treatment. I am glad that I have found someone using the same sonicator. :D I always did want to try higher power but was afraid that the samples will get too hot. I am going to have some more cells coming Monday and will try just sonication as you suggested. Thanks for the info. Hopefully, i will be able to make it work. :D




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