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Breaking nuclear membrane with needle is sufficient (no sonicator nor dounce)?


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#1 crom80

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Posted 03 October 2013 - 07:21 AM

Hi,

 

I am planning to perform a ChIP assay using an Enzymatic ChIP kit from cell signaling (#9002).

 

In short, the kit uses micrococcal nuclease instead of a sonicator to digest DNA.

After digestion, the protocol instructs to either use a sonicator or a dounce to break the nuclear membrane to release cross-linked chromatin.

 

My question is, from your experiences, would running the lysates through a 26 or 27 guage needle about 10 times be as efficient as a dounce to sheer nuclear membrane? Also, are there any precautions that I may have overlooked when using a needle to sheer nuclear membrane?

 

The sample of interest is a very slow growing cell line and will barely grow enough cells (recommended 4x10^7) for an experiment and trying to avoid testing different sheering methods.

 

Thank you.



#2 Curtis

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Posted 03 October 2013 - 07:44 AM

I've done it and it was very inefficient. I did more than 10 times. just don't forget to use the right buffer with recommended pH.



#3 crom80

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Posted 03 October 2013 - 07:58 AM

I've done it and it was very inefficient. I did more than 10 times. just don't forget to use the right buffer with recommended pH.

 

Thank you for your insight. The kit comes with all the buffers so I should be ok.






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