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- W1593594564 abstract "Publisher Summary This chapter discusses the isolation of nuclei suitable for in vitro transcriptional studies. The isolation of intact nuclei from plant sources has been used for a variety of experimental purposes, including the isolation of high-molecular-weight DNA, in vitro DNA synthesis, isolation of labeled transcripts for differential screening of cDNA libraries, preparation of nuclear extracts for in vitro transcription system isolation of nuclear proteins, and studies of protein targeting to the nucleus. The basic steps involved in the process of nuclei isolation include (1) tissue disruption and breaking of cell walls, (2) separation of the nuclei from the crude cellular lysate by filtration, (3) differential centrifugation, (4) solubilization of the plasma membrane and other subcellular organelles with nonionic detergents, and (5) the purification of the intact nuclei from cellular contaminants by continuous or discontinuous density gradient centrifugation. One of the most important uses of isolated nuclei, however, involves the in vitro transcription run-on assay. The accumulation of mRNA can be determined either by increased rates of transcription or by decreased rates of degradation. To distinguish between these two possibilities, in vitro transcription run-on techniques using isolated nuclei were developed by McKnight and Palmiter to measure directly transcription rates from specific genes." @default.
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- W1593594564 date "1995-01-01" @default.
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- W1593594564 title "Chapter 9 Isolation of Nuclei Suitable for in Vitro Transcriptional Studies" @default.
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- W1593594564 doi "https://doi.org/10.1016/s0091-679x(08)61026-2" @default.
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