Applications of Chimeric Genes and Hybrid Proteins Part A: by Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N.

By Melvin I. Simon, Jeremy Thorner, Scott D. Emr, John N. Abelson

The severely acclaimed laboratory usual for greater than 40 years, Methods 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, usually consulted, and praised by means of researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless proper today--truly a necessary book for researchers in all fields of lifestyles sciences

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ColladoVides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao, Science 277, 1453 (1997). tl R. K. Saiki, S. 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.

17O. Huisman and N. Kleckner, Genetics 116, 185 (1987). [21 CONSTRUCTING l a c FUSIONS IN E . c o l i 25 TABLE III A p l a c M u VECTORS FOR MAKING RANDOM FUSIONS Vector Marker ~ Fusion b Fusion type AplacMu50 a AplacMu51 d AplacMu53 d AplacMul I AplacMu3f AplacMu9 j immA imm21 immA, Kan R immA imm21 irnmA, Kan R IacZYA ' lacZYA ' lacZYA ' lac "ZYA' lac "ZYA' lac "ZYA' Transcriptional Transcriptional Transcriptional Translational Translational Translational MuA derivative ~ AplacMu52" AplacMu54 ~" AplacMu55 " AplacMu5" AplacMul 3" AplacMul5" '~imm, phage immunity; Kan R, kanamycin resistance.

Immediately spread the top agar evenly by tilting the plates gently from side to side. Optional step: Prior to the addition of the top agar, the cells and phage may be mixed with prewarmed (37 °) LB broth. This yields bigger plaques, but the surface of the plates can be uneven, and the resulting plates are much sloppier. 5. Allow the plates to cool at room temperature (until the top agar has solidified) and then incubate the plates at 37 ° overnight, agar side down. [2] CONSTRUCTINGlac FUSIONSIN E.

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