{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T18:21:30Z","timestamp":1648837290298},"reference-count":2,"publisher":"Cold Spring Harbor Laboratory","issue":"8","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cold Spring Harb Protoc"],"published-print":{"date-parts":[[2014,8]]},"abstract":"<jats:p>This protocol describes how to generate and analyze products and intermediates in a pre-mRNA splicing reaction. The reaction relies on the use of labeled, capped, synthetic pre-mRNAs, prepared by in vitro transcription, and <jats:italic>Drosophila<\/jats:italic> Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1\u20132 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea\u2013acrylamide gel. Visualization of the splicing reactions will reveal the pre-mRNA, the spliced mRNA, and the intermediates generated by the first step of splicing. For inefficient reactions, a more sensitive detection method, such as RNase protection, primer extension, or RT-PCR (reverse transcription\u2013polymerase chain reaction), may be required.<\/jats:p>","DOI":"10.1101\/pdb.prot080853","type":"journal-article","created":{"date-parts":[[2014,8,4]],"date-time":"2014-08-04T09:13:57Z","timestamp":1407143637000},"page":"pdb.prot080853","source":"Crossref","is-referenced-by-count":1,"title":["In Vitro Splicing Reactions in <i>Drosophila<\/i> Kc Nuclear Extracts"],"prefix":"10.1101","volume":"2014","author":[{"given":"Donald C.","family":"Rio","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"246","published-online":{"date-parts":[[2014,8,1]]},"reference":[{"key":"2021111008554335000_2014.8.pdb.prot080853.1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.85.9.2904"},{"key":"2021111008554335000_2014.8.pdb.prot080853.2","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot080846"}],"container-title":["Cold Spring Harbor Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/pdb.prot080853","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,10]],"date-time":"2021-11-10T19:11:38Z","timestamp":1636571498000},"score":1,"resource":{"primary":{"URL":"http:\/\/www.cshprotocols.org\/lookup\/doi\/10.1101\/pdb.prot080853"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,8]]},"references-count":2,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2014,8,1]]},"published-print":{"date-parts":[[2014,8]]}},"alternative-id":["10.1101\/pdb.prot080853"],"URL":"https:\/\/doi.org\/10.1101\/pdb.prot080853","relation":{},"ISSN":["1940-3402","1559-6095"],"issn-type":[{"value":"1940-3402","type":"print"},{"value":"1559-6095","type":"electronic"}],"subject":[],"published":{"date-parts":[[2014,8]]}}}