In vitro transcription by RNA polymerase II in extracts of Xenopus oocytes, eggs, and somatic cells.
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 mM KCl, 12 mM MgCl2), template concentration (20-40 micrograms/ml), incubation time (30-60 min), and temperature (25 degrees C) for class II gene transcription are described.
Toyoda
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Laboratory of Molecular Embryology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Wolffe
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AP
D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts; D002478:Cells, Cultured; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D013696:Temperature; D014158:Transcription, Genetic; D014982:Xenopus laevis
D002457:Cell Extracts; D012319:RNA Polymerase II