By John M. Walker
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4. 5. 6. 7. 8. Clemens, M. J. D. and Higgins, S. ) IRL Press, Oxford and Washington. 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.
Prepare fresh hybridization buffer and warm to 42OC. 5 mL of hybridization buffer at 42OC. 2. Blot dry on a clean filter paper, and place the DNA papers in a sterile, siliconized bijou bottle. 3. Prepare a solution of RNA in hybridization buffer. 2 mg/mL poly (A)+ RNA in sterile distilled water (seeNote 4 in section 4). 4,20 mM EDTA, 10 PL of 10% (w/v) SDS, 250 PL of deionized formamide, and 75 PL of 4M NaCl, to give a final volume of 500 PL (seeNote 5 in section 4). 4. Apply the RNA solution to the DNA papers, seal the bottle tightly, and incubate at 42OCfor 16 h (seeNote 6 in section 4).
8. 9, Paterson, B. , Roberts, B. , and Kuff, E. , (1977) Structural gene identification and mapping by DNAmRNA hybrid-arrested cell free translation. Proc. Nufl. Ad. Sci USA 74,43704374. Anderson, M. L. M. and Young, B. D. (1985) Quantitative Filter Hybridization, in Nucleic Acid Hybridization-A Practical Approach (Hames, B. D. and Higgins, S. ) IRL, Oxford. Forde, B. G. M. ) MacMillan, New York. Slater, R. J. M. ) MacMillan, New York. Odell, J. T. and Howell, S. H. (1980) The identification, mapping and characterisation of mRNA for p66 a cauliflower mosaic virus encoded protein.