Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries

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dc.contributor.author Kurnit, David M.
dc.date.accessioned 2020-01-13T15:21:36Z
dc.date.available 2020-01-13T15:21:36Z
dc.date.issued 1994-07
dc.identifier.citation Kurnit, David M. (1994). "Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries." Current Protocols in Molecular Biology 27(1): 6.12.1-6.12.12.
dc.identifier.issn 1934-3639
dc.identifier.issn 1934-3647
dc.identifier.uri http://hdl.handle.net/2027.42/153261
dc.description.abstract The recombination‐based assay represents a convenient way to screen a complex library constructed in bacteriophage l for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 106 to 107 plaque‐forming units (pfu) may be screened using several petri dishes, and the homology for crossing‐over need only be greater than 25 bp, the RBA represents an efficient way to screen complex l libraries rapidly for homology to a given sequence. In this procedure, a l library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross‐over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of l on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents l propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the l vector.
dc.publisher John Wiley & Sons
dc.title Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries
dc.type Article
dc.rights.robots IndexNoFollow
dc.subject.hlbsecondlevel Molecular, Cellular and Developmental Biology
dc.subject.hlbsecondlevel Genetics
dc.subject.hlbtoplevel Health Sciences
dc.subject.hlbtoplevel Science
dc.description.peerreviewed Peer Reviewed
dc.description.bitstreamurl https://deepblue.lib.umich.edu/bitstream/2027.42/153261/1/cpmb0612.pdf
dc.identifier.doi 10.1002/0471142727.mb0612s27
dc.identifier.source Current Protocols in Molecular Biology
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dc.owningcollname Interdisciplinary and Peer-Reviewed
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