Protocol describes how to produce a soluble nuclear extract rich in basal pol II transcription factors from Drosophila embryos. This is a cell-free extract that contains all the necessary transcription factors and is capable of accurate initiation of transcription by RNA polymerase II but is deficient in core histones and histone H1. - [Read Preparation of a Highly Efficient Transcription Extract from Drosophila Embryos Protocol]
In this protocol nuclei isolated from cells expressing the gene of interest are incubated with radiolabeled UTP which is incorporated into nascent RNA transcripts by RNA polymerase molecules that were actively transcribing at the time the cells were harvested. Because very little denovo initiation of RNA synthesis occurs in isolated nuclei, transcription of the target gene can be measured by hybridizing the radiolabeled RNA to an excess of the target gene immobilized on a nitrocellulose or nylon - [Read Protocol for Transcriptional Run- On Assays]