In Vitro Splicing Reactions in Drosophila Kc Nuclear Extracts

Abstract

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 Drosophila Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1–2 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea–acrylamide 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–polymerase chain reaction), may be required.