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By John N. Abelson, Melvin I. Simon, Ray Wu

The significantly acclaimed laboratory usual, tools in Enzymology, is likely one of the such a lot hugely revered courses within the box of biochemistry. considering that 1955, each one quantity has been eagerly awaited, often consulted, and praised by way of researchers and reviewers alike. The sequence includes a lot fabric nonetheless proper today--truly a necessary ebook for researchers in all fields of lifestyles sciences.

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Sci. A. 85, 1595 (1985). ~7 j. R. McLachlin, M. A. Eglitis, K. Ueda, P. W. Kantoff, I. H. Pastan, W. F. Anderson, and M. M. Gottesman, J. Natl. Cancer Inst. 82, 1260 (1990). ~8 U. A. Germann, M. M. Gottesman, and I. Pastan, J. Biol. Chem. 2,64, 7418 (1989). 19 U. A. -V. Chin, I. Pastan, and M. M. Gottesman, FASEB J. 4, 1501 (1990). 38 VECTORS FOR EXPRESSING CLONED GENES [4] genes, MDR1 and the heterologous gene, on a polycistronic messenger RNA or by including a separate promoter plus foreign sequences inside the LTRs.

Larsen, and R. M. Kay, Mol. Cell. Biol. 5, 1750 (1985). 16 G. N. Pavlakis and D. Hamer, Proc. Natl. Acad. Sci. A. 80, 397 (1983). 17 D. R. Hurwitz, R. Hodges, W. Drohan, and N. Sarver, Nucleic Acids Res. 15, 7137 (1987). [5] HIGH-LEVEL GENE EXPRESSION BY T7 R N A POLYMERASE 49 known. ~8-2° The latter, in turn, are so different in sequence from bacterial promoters that they cannot be transcribed by the bacterial RNA polymerase. ~8Moss and co-workers 2~'20were first in using this principle for the short-term vaccinia expression system.

Tubes are kept wrapped in foil and frozen at - 2 0 °. 5% (v/v). 3. Colonies will appear by 7-14 days after selection is initiated (again, this should vary with cell type). 5% (w/v) methylene blue in 50% (v/v) ethanol. Let sit for 5 min on the cells and wash off with several rinses of H20. Background will appear light blue because drug-sensitive cells are not killed by colchicine but are prevented from dividing. ~5 Viral titers of approximately 8 x 104 transforming units per milliliter of supernatant have been obtained.

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