Mammalian In Vitro Splicing Assays

  • Akila Mayeda
  • Adrian R. Krainer
Part of the Methods in Molecular Biology™ book series (MIMB, volume 118)

Abstract

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.

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Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Akila Mayeda
    • 1
  • Adrian R. Krainer
    • 2
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Miami School of MedicineMiamiUSA
  2. 2.Cold Spring Harbor LaboratoryCold Spring HarborUSA

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