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cytoplasm and nuclear run off


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#1 herbie

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Posted 13 January 2011 - 07:54 AM

I want to seperate nucleus from cytoplasm and pool the cytoplasm, transfect a plasmid to cytoplasm and then add nucleus to demonstrate transcription. Has anyone done a simialr work and if so how did you go about seperating and pooling the nucleus and cytoplasm

Thank you,

Herbie

#2 bob1

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Posted 13 January 2011 - 02:23 PM

So basically doing a sub-cellular fractionation and then adding nuclei? You are aware that this will destroy the cells unless you are planning on removing the nuclei individually from the cells by patch-clamping style techniques?

#3 herbie

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Posted 14 January 2011 - 01:59 AM

So basically doing a sub-cellular fractionation and then adding nuclei? You are aware that this will destroy the cells unless you are planning on removing the nuclei individually from the cells by patch-clamping style techniques?


Thank you for the reply, I figured it would. How do I go about to remove the nucleus individually - sub-cellular fractionation

#4 bob1

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Posted 15 January 2011 - 06:37 PM

You can isolate intact nuclei by sub-cellular fractionation. If you want viable cells out the end, you will need to investigate techniques that remove the nucleus by micromanipulation (look for protocols around cloning of animals - most people do this with animal eggs).




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