Small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing

https://doi.org/10.1016/0076-6879(90)81108-7Get rights and content

Publisher Summary

This chapter focuses on small-scale preparation of extracts from radiolabeled cells efficient in pre-mRNA splicing. By several criteria, nuclear extracts prepared by the small-scale procedure are comparable to extracts prepared by the conventional procedure. Miniextracts are fully active in pre-mRNA splicing and also efficiently transcribe class II promoters. Harvested cells are resuspended in a volume of buffer A equal to the packed cell volume, left to swell on ice for 15 rain, and then lysed with 30 strokes of a B-type Dounce homogenizer. The short dialysis period results in less precipitation of denatured protein and is sufficient to adjust the extract to the desired ionic conditions. Precipitate that forms during dialysis can be removed by centrifugation of the extract for 10 min at 2000 rpm in a Beckman J6B centrifuge using a JS 4.2 rotor. Monolayer cells at 80% confluence are harvested using a rubber policeman or by trypsinization. Cells are washed in 30 volumes of phosphate-buffered saline (PBS) and the packed cell volume determined by pelleting for 5 minutes at 1200 rpm in a J6B centrifuge using a JS 4.2 rotor. Packed cells are resuspended in one packed cell volume of buffer A and allowed to swell on ice for 15 min.

References (18)

  • A.R. Krainer et al.

    Cell

    (1984)
  • N. Hernandez et al.

    Cell

    (1983)
  • K.A.W. Lee et al.

    Gene. Anal. Tech.

    (1988)
  • B. Ruskin et al.

    Cell

    (1984)
  • D. Frendewey et al.

    Cell

    (1985)
  • P.J. Grabowski et al.

    Cell

    (1985)
  • A.R. Krainer et al.

    Cell

    (1985)
  • D.L. Black et al.

    Cell

    (1985)
  • D.L. Black et al.

    Cell

    (1986)
There are more references available in the full text version of this article.

Cited by (0)

View full text