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By John M. Walker

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2. 3. 4. Place the gel slice containing the DNA of interest into a tube. Add approximately 10 vol of low-salt buffer 1. Heat the sample with occasional shaking at 65OC for approximately 30 min. Ensure that the agarose is completely me1ted. Place the DNA sample and low-salt buffer in a 42OCwater bath (Note 8 in section 4). 1, but use prewarmed (42OC) low-salt buffer in all steps.

If the experiment is designed to show that a particular cDNA clone codes for a particular translation product, deciding the amount of RNA is not a problem. It is not normally necessary to purify poly (A)+ RNA for hybrid arrested translation. In cases in which the expression of a messenger RNA is particularly low, this may be desirable, but I have found that by using total cellular RNA the messenger RNA is protected from degradation by any ribonuclease that may have entered the system. The basis for this is uncertain, but it may be that the hugeexcess of ribosomal RNA saturates any ribonuclease.

Pain, V. M. (1986) Initiation of protein synthesis in mammalian cells. J. 235‘625637. Pain, V. , Lewis, J. ,Hen&w, E. , and Clemens, M. (1980) The effects of amino acid starvation on regulation of polypeptide chain initiation in Ehrlich ascites tumor cells. 1. Bid. Chem. 255, 1486-1491. J. and Jackson,R. J. (1985) Preparation and properties of an improved cell-free protein synthesis system from mammalian liver. Actu 825,45-56. Austin, S. , Pollard, J. , and Clemens, M. J. (1986) Regulation of polypeptide chain initiation and activity of initiation factor eIF-2 in Chinese-hamster ovary cell mutants containing temperature-sensitive aminoacyl-tRNA synthetases.

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