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Extra info for Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification

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Note: After 6 hr, add chloroform, irrespective of whether there is obvious lysis (occasionally, high-titer lysates can be obtained without apparent lysis). 9. Vortex the tubes briefly and then centrifuge at 4500g for 10 min. 10. Transfer the supernatant to sterile screw-cap tubes (taking care to avoid transferring any of the chloroform) and store in the dark at 4 °. , they have picked up the plasmid encoded lac fusion) and some of which are nonrecombinant Lac- parental phage. The next step in the procedure is to isolate recombinant Lac + plaques and use them to prepare high-titer phage lysates.

Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, Science 230, 1350 (1985). ~2R. F. Moreira and C. J. Noren, Biotechniques 19, 56, 58 (1995). under the control of the strong, inducible placUV5 promoter. Recombination of fusions with low activity onto these A vectors can therefore be detected easily against a background of dark blue plaques. The fact that the inserts in ARS45 and ARS74 are in the opposite orientation with respect to those in ARS88 and ARS91 is not relevant.

Tetrazolium is a less sensitive indicator medium than MacConkey, requiring both a functional l a c y + gene and approximately 400 to 500 Miller units of/3-galactosidase activity for the formation of Lac + (white) colonies. Somewhat paradoxically, it is precisely this insensitivity that makes tetrazolium such a useful indicator. Tetrazolium agar can be used to screen for rare mutations that decrease the/3-galactosidase activity of a fusion with high activity. Differences between strains with 300 Miller units of /3-galactosidase activity and those with wild-type (1000 Miller units) activity are indistinguishable on minimal lactose or X-gal agar and are very difficult to detect on lactose MacConkey agar.

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