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Nu-cyto separation


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#1 Cheng

Cheng

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Posted 02 November 2010 - 12:41 AM

Hi,
i truly appreciate if anyone could give any comments or suggestions here.
I am trying to separate cyto and nuclear fraction and then extract RNA for analysis.
Initially we were using a kit to do the seperation, but eversince we tried to change to another cell line (F10 cells), we find that the kit (PARIS, Ambion) that we were using could not work on this new cell line.
Thus, i tried to adapt a labmate's protocol to fit my experiment.

The protocol uses hypotonic lysis buffer to break cell membrane with the following materials.
KCl (1.5mM)
MgCl2 (2.5mM)
Tris-HCl pH7.4 (5mM)
Triton X-100 (1%)
Sodium Deoxycholate (1%)
DTT (1mM)
RNase inhibitor (100U/ml)

My friend uses this for polysome analysis and it's fine for him to stop here as he only needs cytoplasm extract, but now i want to modify the above materials to achieve hypertonic solution to break nuclear membrane as well if it's possible to think this way.

Anyone has suggestions, or nu-cyto fractionation protocol that is suitable for RNA downstream assays, feel free to write.
thank you very much!




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