By Thorner J. (ed.), Emr S.D. (ed.), Abelson J.N. (ed.)
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A. Federspiel, and S. Falkow,Science288, 1436 (2000). 5, 1 mM EDTA). Vector Preparation. Digest 1 txg of pFPV25 or pFPV27 with 2 U of BamHI at 37° for 1 hr (100 tzl total volume). Add 2 U of calf intestine alkaline phosphatase (CIAP) to the digestion mixture and incubate at 37° for an additional hour. Heat inactivate the CIAP at 65° for 15 min and extract the digestion mixture twice with TE-saturated phenol/choloroform (1:1, v/v) to remove any residual BamHI and CIAP activity. Ethanol precipitate the DNA and resuspend in 20/zl of TE.
4 R. H. Valdivia, B. P. Cormack, and S. Falkow, in "GFP: Green Fluorescent Protein Strategies and Applications" (M. Chalfie and S. ). Wiley-Liss, New York, 1998. 5 M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward, and D. C. Prasher, Science 263, 802 (1994). 6 A. B. , TIBS 20, 448 (1995). 7 B. P. Cormack, R. H. Valdivia, and S. Falkow, Gene 173, 33 (1996). D. R. Parks, L. A. Herzenberg, and L. A. Herzenberg, in "Fundamental Immunology" (W. E. ), p. 781. Raven Press, New York, 1989. 9 R. H. Valdivia and S.
Microbiol. 47, 945 (1993). E. U. S. ~. I U . 40\ , -I" . . . 76 :Y7 kb 'phoA IoxP • IoxP FIG. 1. Transposons for generating alkaline phosphatase gene fusions and in frame epitope insertions. phoA, alkaline phosphatase gene; cat, chloramphenicol acetyltransferase gene; IoxP, site-specific recombination sequence. (ISphoA/in) may be used to generate phoA fusions to cloned genes, which may then be converted in vitro into 31 codon insertion mutations (Fig. 2). 15 The second transposon (ISphoA/hah) may be used to generate fusions to chromosomal genes (and cloned genes), which may be converted in vivo into 63 codon insertions (Fig.
Applications of Chimeric Genes and Hybrid Proteins by Thorner J. (ed.), Emr S.D. (ed.), Abelson J.N. (ed.)