|MadSci Network: Molecular Biology|
Dear Jennie, your question is very valuable. First of all, let's clarify some terms. The word "nucleus extraction" refers to the isolation of the DNA inside the cell. In your case, you are after the nucleus itself. Thus, the expression "nucleus isolation" is more appropriate. The main procedure is usually achieved by employing cell lysis by gentle mechanical means or with mild detergents, followed by fractionation of cellular components by differential centrifugation. There are several standard methods for isolating nuclei from mammalian cells. The resulting nuclei are functional for the synthesis and extension of endogenous RNA primary transcripts. The method for nuclei isolation from tissue culture cells utilizes a detergent that breaks the cell membrane, releasing all the content of the cell. Since nuclei are the largest organelles in the cell, they are easily separated form other organelles in the cell by low speed centrifugation and further purified by repeated washes in the same lysis buffer. For isolation of nuclei from solid tissues or from cell lines with fragile nuclei, the nuclei are purified by centrifugation through a dense sucrose cushion to protect nuclei and strip away cytoplasmic contaminants. As an example, here is a short protocol: Cells are resuspended in a buffer containing sucrose and a detergent (Triton). Then, the cells are gently vortexed for a short time and allowed to incubate on ice for a few minutes. Nuclei are centrifuged and washed twice with the same buffer. That is basically it. There is several ways to check the nuclei integrity and the yield of isolation. Lastly, the company Sigma-Aldrich now sells a kit for the isolation of nuclei (Nuclei EZ Prep Nuclei Isolation Kit, Product Code: NUC-101). Check out this web page for a description: http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Life_Science_Quarterly/July_2000/July_2000_Cell_Biology.html Hope this helps, Daniel
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