Richard A. Young

17 – λgt 11: Gene Isolation with Antibody Probes and Other Applications

Publisher Summary This chapter discusses the methods for the isolation of eukaryotic and prokaryotic genes by screening Escherichia coli expression libraries with antibody probes using the bacteriophage expression vector λgt11. For the λgt 11 fusions examined thus far, the intact fusion proteins comprise from less than 0.1% up to 4% of the total E. coli protein. The E. coli host, Y1090, has three features that are useful for screening λgt 11 expression libraries with antibody probes. In genomic DNA libraries, the probability of having a particular gene fused to lacZ depends on the length of the gene and the size of the organisms genome. Successful immunoscreening depends on the quality of the antibody. The recombinant subclones are characterized further to ascertain whether they contain multiple inserts or rearranged insert DNA, either of which could complicate the interpretation of the data.