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 (seeNote 1 ). The pre-mRNA substrates are usually prepared by in vitro runoff transcription with a bacteriophage polymerase (seeChapter 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 onref.1 , with modifications introduced inrefs.2 ,3 , and have been routinely used in our laboratory for many years.
