In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells

Abstract

We describe procedures for preparing extracts of Xenopus oocytes, eggs, and somatic cells that will accurately transcribe class II genes. A variety of viral and Xenopus promoters direct the accurate initiation of transcription by RNA polymerase II in these extracts. Optimal ionic conditions (100–200 m m KCl, 12 m m MgCl 2), template concentration (20–40 μg/ml), incubation time (30–60 min), and temperature (25°C) for class II gene transcription are described.