Nuclei Isolation Kit: Nuclei PURE Prep 

For preparation of pure nuclei and fragile nuclei from cell lines and solid tissues.

The protocol incorporates centrifugation through a dense sucrose cushion to protect nuclei and strip away cytoplasmic contaminants. The sucrose concentration that is suitable for a particular cell type is determined empirically by the user. The sucrose concentrate and sucrose cushion buffer give the user flexibility to modify the density of the sucrose cushion as appropriate. High yield has been obtained from common cell lines (Jurkat, HFN7.1, COS7, HEK293 and MDCK) and tissues (spleen and liver). These preparations are suitable for many cell biology applications, e.g., as a source of nuclear components such as chromatin, genomic DNA, histones, and nuclear RNA/RNP, produces nuclei for in vitro apoptosis assays, and functional studies such as examination of the transcriptional status of cells.

The protocol incorporates centrifugation through a dense sucrose cushion to protect nuclei and strip away cytoplasmic contaminants. High yield has been obtained from common cell lines (Jurkat, HFN7.1, COS7, HEK293 and MDCK) and tissues (spleen and liver). These preparations are suitable for many cell biology applications, e.g., as a source of nuclear components such as chromatin, genomic DNA, histones, and nuclear RNA/RNP, produces nuclei for in vitro apoptosis assays, and functional studies such as examination of the transcriptional status of cells.

sufficient for 15 nuclei preparations (~1-10×10⁷ cells or 1g of tissue per preparation)

nuclease and protease, free

wet ice

2-8°C

, Robert E. Farrell, Jr., ed., Analysis of nuclear RNA, Chapter 14. RNA Methodologies: A Laboratory Guide for Isolation and Characterization San Diego,CA, , (1993), 235-263

Blobel, G., and Potter, V.R., Nuclei from rat liver: isolation method that combines purity with high yield. Science 154, 1662-1665, (1966) abstract

Greenberg, M.E., and Bender, T.P., et al., Ausubel, F.M., ed., Identification of newly transcribed RNA. Current Protocols in Molecular Biology New York, NY , (1987), 4.10.1-4.10.11

Marzluff, W.F., and Huang, R.C.C., Hames, B.D., and Higgens, S.J., ed., Transcription of RNA in isolated nuclei, Chapter 4. Transcription and Translation: A Practical Approach,, (1984), 89-129

Nuclei PURE Lysis Buffer 180 mL

Nuclei PURE Storage Buffer 90 mL

10% Triton X-100 Solution 1.7 mL