UNIT 6.12 Recombination-Based Assay (RBA) for Screening Bacteriophage Lambda Libraries
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Kurnit, D. M. 2001. Recombination-Based Assay (RBA) for Screening Bacteriophage Lambda Libraries. Current Protocols in Molecular Biology. 27:VI:6.12:6.12.1–6.12.12.
- Published Online: 1 MAY 2001
- Published Print: JUL 1994
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.