A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing.
A convenient and rapid method for preparing soluble extracts from the nuclei of as few as 3 x 10(7) mammalian cells (miniextract procedure) is described. By several criteria, miniextracts are comparable to nuclear extracts prepared from large numbers of cells by the conventional procedure. Miniextracts are able to support efficient transcription of a variety of class II promoters. In addition, DNase I footprinting and gel retardation assays can be performed directly in miniextracts, enabling the detection of sequence-specific DNA-binding proteins. Besides transcription, miniextracts efficiently carry out pre-mRNA splicing and allow formation and fractionation of previously characterized splicing complexes. The small-scale procedure enables simultaneous preparation of multiple extracts from a variety of cell types under different experimental conditions. Moreover, the use of small amounts of cells allows minimal expenditure of valuable or expensive materials such as radioactive compounds. Consequently, the procedure is highly advantageous for biochemical analysis of transcription and RNA processing in mammalian cells.
Lee
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KA
Harvard University, Cambridge, Massachusetts 02138.
Bindereif
A
A
Green
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MR
D004268:DNA-Binding Proteins / Q000032:analysis; D003850:Deoxyribonuclease I / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009710:Nucleotide Mapping; D010957:Plasmids; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I