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Nonaqueous isolation of transcriptionally active nuclei fromXenopusoocytes

Elsevier Science & Technology
DOI: 10.1016/0076-6879(90)81110-g
  • Section I. Processing Of Messenger Rnas A. Mammalian Cell Extracts And Assays


Publisher Summary This chapter describes methods for using the oil-isolated nucleus (OIN) to examine RNA transcription, processing, and transport. The puncture method for isolating the nucleus under oil is derived from the long-utilized technique for manual isolation of Xenopus oocyte nuclei (germinal vesicles) into aqueous solutions. First, individual oocytes are dissected from the ovary under extracellular medium. Because the oocyte is blotted prior to puncture, the nucleus passes from the cell into the oil without being exposed to an aqueous phase. After the nucleus is extruded, the cell is gently shaken to free the nucleus from the pedestal of cytoplasm which emerges after it. The nucleus, covered by a layer of cytoplasm, protrudes through the tear and can be separated from the remainder of the cell using the tips of the forceps or a similar microtool. Cytoplasm is not stripped from the nucleus by passage through a size-restricted hole as in the puncture method.

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