3 replies to this topic

[herbie]

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

[bob1]

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?

[herbie]

bob1, on 13 January 2011 - 02:23 PM, said:

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

[bob1]

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).