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Resistance to shearing in sonication?..


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#1 Maayan

Maayan

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Posted 28 May 2014 - 08:54 AM

Hi all, 

 

I've been having some major difficulties with the sonication part of the ChIP. I am working on mice brain tissue (PVN punches), crossed-linked in fresh formaldehyde (diluted in sterile PBS) and lysate with 1% SDS buffer. For some reason, which I hope you will help me reveal, I always get 1500 bp fragments, no matter how long I sonicate the sample. On the gel the DNA appear to reach the desired size- 500-200bp (fig. A), but the TapeStation analysis show otherwise (fig. B.)

 

Screen Shot 2014-05-28 at 7.49.30 PM.png

Screen Shot 2014-05-28 at 7.49.36 PM.png

 

I have tried so many things to solve this..

 

I am using a Branson 450 sonicator, but I've also tried a different brand; I have tried calibration of the amplitude, different durations of On/Off pulse, different concentrations of formaldehyde and several fixation durations. 

 

And if you're still with me, here's the protocol I'm using:

 

1. Punching the brain and placing in 4/2% fresh formaldehyde (diluted in sterile PBS) for 10' followed by termination of fixation with 0.125 glycine for 5', and 3 washes with 1x PBS. All solutions contain protease inhibitors. Finally the samples are flash frozen in liquid nitrogen.

2. Before sonication, I lysate the tissue with cell lysis buffer on ice for 20' followed by nuclei lysis buffer on ice for 20'. 

3. Sonication parameters: 20/30/40/45% amplitude; 0.5 sec On, 1.5 sec Off; 30 seconds- 6 minutes "On time"in total; placing on ice for 2 minutes every 30 seconds. Following the sonication I treat the DNA with RNase A. 

 

Many thanks in advance,

Desperado. 

 

 

 



#2 trungbiotech

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Posted 01 June 2014 - 08:35 PM

Perhaps you can try MNase digestion prior to sonication






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