In Vitro Translation

  • Rod J. Scott
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 49)

Abstract

In vitro translation remains a powerful technique for the identification, characterization, and quantification of mRNAs from a wide range of sources. The technique is also useful for assessing the quality of mRNA prior to cDNA synthesis. There are numerous in vitro translation systems, but among the most useful are derived from wheat germ (1, 2) and rabbit reticulocytes (3,4). The rabbit reticulocyte system is probably the more efficient of the two, particularly in translating larger mRNAs. However, the wheat germ system does offer a genuine “animal-friendly” alternative. Both systems are commercially available as well defined and highly active preparations, and are therefore the most convenient way of carrying out in vitro translations. However, for those with sufficient incentive, the wheat germ system is relatively easy to prepare and the reader is referred to Speirs (5).

Keywords

Vortex Toluene Electrophoresis Methionine Fluor 

References

  1. 1.
    Roberts, B. E. and Paterson, B. M. (1973) Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc. Natl. Acad Sci. USA 70, 233–2334.CrossRefGoogle Scholar
  2. 2.
    Marcu, K. and Dudock, B. (1974) Characterisation of a highly efficient protein synthesizing system derived from commercial wheat germ. Nucleic Acids Res. 1, 1385–1397.PubMedCrossRefGoogle Scholar
  3. 3.
    Schimke, R. T., Rhoads, R. E., and McKnight, S. (1974) Assay of ovalbumin mRNA in reticulocyte lysate. Meth. Enzymol 30, 694–701.PubMedCrossRefGoogle Scholar
  4. 4.
    Pelham, H. R. B. and Jackson, R. J. (1976) An efficient mRNA-dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67, 247–256.PubMedCrossRefGoogle Scholar
  5. 5.
    Speirs, J. (1993) In vitro translation of plant messenger RNA. Methods in Plant Biochemistry, vol 10 (Bryant, J., ed.), Academic, London, pp. 33–56.Google Scholar
  6. 6.
    Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of the head of Bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  7. 7.
    Laskey, R. A. and Mills, A. D. (1975) Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335–341.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1995

Authors and Affiliations

  • Rod J. Scott
    • 1
  1. 1.Department of BotanyUniversity of LeicesterUK

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