Mammalian in vitro splicing assays

Mayeda, A., Krainer, A. R. (1999) Mammalian in vitro splicing assays. Methods in Molecular Biology, 118. pp. 315-21. ISSN 1064-3745 (Print)1064-3745

DOI: 10.1385/1-59259-676-2:315


Splicing reactions are typically carried out using nuclear extracts, S100 extracts complemented with SR proteins, or partially purified fractions derived from the crude extracts. The extract preparation procedures are described in Chapter 24. Extracts derived from HeLa cells are used most commonly (see Note 1). The pre-mRNA substrates are usually prepared by in vitro runoff transcription with a bacteriophage polymerase (see Chapter 1). The intermediates and products of splicing are most conveniently visualized by urea/polyacrylamide gel electrophoresis (urea-PAGE) and autoradiography, which requires the use of labeled pre-mRNA substrate. The protocols provided here are based on ref. 1, with modifications introduced in refs. 2,3, and have been routinely used in our laboratory for many years.

Item Type: Paper
Uncontrolled Keywords: Cell Extracts/chemistry Cell Nucleus/chemistry Electrophoresis, Polyacrylamide Gel HeLa Cells Humans Nuclear Proteins/isolation & purification Plasmids RNA Precursors/isolation & purification RNA Splicing/*genetics Transcription, Genetic
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA splicing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > splicing factor
CSHL Authors:
Communities: CSHL labs > Krainer lab
Depositing User: Matt Covey
Date: 1999
Date Deposited: 11 Mar 2014 21:06
Last Modified: 11 Mar 2014 21:06
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