Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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Altered -Lactamase Selection Approach for Site-Directed Mutagenesis

(Protocol summary only for purposes of this preview site)

Many protocols exist to perform site-directed mutagenesis, and here we present one of the more commonly used onessite-directed mutagenesis by altered -lactamase selection. -Lactamase is an enzyme that cleaves ampicillin, rendering it impotent to bacteria. Certain mutations in the active site of -lactamase can alter the substrate specificity of the enzyme and allow it to have increased hydrolytic activity for the cephalosporin family of antibiotics, a property not shared by wild-type lactamases (Cantu et al. 1996). E. coli cells carrying the -lactamase triple mutant G238S:E240:R241G show increased resistance to cefotaxime and ceftriaxone, two cephalosporins, compared with wild-type cells. This protocol takes advantage of this property to select for plasmids that have undergone site-directed mutagenesis.


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(Limited time special offer.) Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook
Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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