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Cell lysis before shearing

lysis nuclei nucleus shear

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5 replies to this topic

#1 Wonyong

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Posted 04 September 2011 - 08:13 PM

Hi there,

I am trying to do ChIP on mouse CD4 T cells, but currently suffering inefficient DNA shearing issue.
I use Millipore's magna-ChIP kit and its protocol mentions to perform cell lysis and nuclei isolation before shearing.
Since having an issue with shearing, I checked with microscope if cells were lysed well after the lysis step. And the result was they were all intact even after several swelling and lysis atempts.
My protocol uses 15 minutes of cell swelling with a buffer containing no NaCl, and additiion of NP-40 final concentration 0.5% and hard vortexing.
How could I solve the issue?
Thank you in advance.

#2 EMD Millipore

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Posted 06 September 2011 - 10:53 AM

Hi -
I work at EMD Millipore, and contacted our Technical Service group about your inquiry. Please see their response below:

In the kit, there is a Cell Lysis Buffer and Nuclear Lysis Buffer. Did you happen to use those in your research? If not, you can improve the lysis efficiency of the isolated nuclei by either multiple freeze/thaws or by a brief sonication (in the nuclear lysis buffer) to assist nuclear lysis.

If you would like more help, please do not hesitate to call our technical service group at 1-800-645-5476.

Thank you -
Diane

#3 Wonyong

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Posted 23 September 2011 - 01:01 AM

Hi Diane,

Thank you for your answer.

Instead of cell lysis buffer in the kit, I use handmade lysis buffer. Its recipe is from a Millipore tech support who told me it would work just the same.
10mM HEPES, pH7.9, 0.5% IGEPAL-CA630, 1.5mM MgCl2, 10mM KCl

I tried your suggestions, multiple freeze and thaw and brief sonication, and unfortunately found no significant differences in neither cell lysis nor DNA shearing performance from no treated sample.

The sample I am trying with is EL4, murine T cell lymphoma cell line, which is a substitute for primary T cell. I could not waste valuable primary cells on DNA shearing test. And from my experience, primary T cells are even tougher to shear than EL4.

Since I live in South Korea, I doubt that reaching your technical service on phone could be an option.

Do you have other suggestions I can try?

#4 chabraha

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Posted 23 September 2011 - 07:45 AM

2 things:

Because your cells are fixed don't expect them to look disrupted (ie- no intact nuclei) until after sonication
Use a 1% SDS lysis buffer for your sonication, then dilute it 1:10 in a buffer containing TritonX-100 for the immunoprecipitaiton step.

Good luck
Treasure Chest Wizardry

#5 Wonyong

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Posted 25 September 2011 - 05:02 PM

chabraha,

Thank you for your answers.
I did not know about the first one. However, cell lysis did not enhance DNA shearing on a gel either.
About the second, I do use 1% SDS lysis buffer for DNA shearing. All the buffers I have mentioned above was for cell lysis, a step before nuclear lysis.

I wonder SDS precipitation could affect DNA shearing efficiency. Every time I store my samples on ice SDS forms white particles.

#6 chabraha

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Posted 26 September 2011 - 07:43 AM

you should warm your SDS lysis buffer to room temp before you add it to your nuclei. Run the sample through a 20guage needle 6-10 times and then sonicate your sample at least 8 times with near 80% power for 3-5 seconds each time
Treasure Chest Wizardry





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