Preparation of Drosophila Kc cell nuclear extracts for in vitro splicing.

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This protocol describes how to prepare nuclear extracts from cultured Drosophila cells. It uses the Kc cell line, which grows without fetal bovine serum and so is suitable for large-scale suspension cultures. The suspension cells are harvested by centrifugation, washed to remove media components, and resuspended in a low-salt (hypotonic) buffer that makes the cells swell in size and easy to homogenize. Usually, nuclear extract preparations are performed on 4-32 L of cells at ∼ 3.5 × 10(6) cells/mL.