Dignam and Roeder Nuclear Extract Preparation
Adapted from Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 2nd edition, by Michael F. Carey, Craig L. Peterson, and Stephen T. Smale. CSHL Press, Cold Spring Harbor, NY, USA, 2009.INTRODUCTION
In this protocol for Dignam and Roeder nuclear extract preparation, HeLa cells are harvested by centrifugation, washed, and resuspended in a hypotonic buffer, which causes the cells to swell. Subsequently, the cells are lysed with a handheld Dounce homogenizer. The nuclei are pelleted by centrifugation, the cytoplasmic supernatant is decanted, and the nuclear pellet is resuspended by Dounce homogenization in a moderate salt buffer. After stirring the suspension for 30 min to allow extraction of transcription factors, the nuclei are centrifuged again, and the resulting supernatant or extract is dialyzed for use in transcription experiments. Generally, 1 L of culture (i.e., 1-2 g of cells, depending on cell density at harvest) yields 1-2 mL of extract at 6-10 mg/mL. This should provide enough extract for ~50-100 in vitro transcription reactions, depending on the scale.
