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A Positive Selection Assay for Mutation Analysis in Big Blue,,,Animals

providing the lacI plaque color-screening assay, Stratagene is now offering an alternative method of screening for mutations in Big Blue rodents, the l Select-cII mutation assay kit. In this positive selection assay, the region encompassing the cII gene of coliphage lambda functions as the mutational test sequence.4 The cII gene encodes a protein that activates transcriptional promoters in lambda that are essential for lysogenization. Mutations in the cII region that lower the levels of cII protein result in a decreased ability of lambda to lysogenize. When grown under conditions that favor lysogeny, lambda prophages carrying such mutations (lcII ) survive only by entering the lytic pathway of development, forming plaques. Prophages that are wild type for the cII region (lcII +) integrate into the host genome and become part of the developing bacterial lawn.

figure 2

The method of performing the l Select-cII mutation assay is outlined (figure 2). After rescuing the lLIZ shuttle vector from the rodent genomic DNA, the packaged virions are used to infect the E. coli host strain G1250 (a specialized hfl version of Stratagenes XL1-Blue MRA cells). The infected host cells are then plated and grown at 24C for 48 hours, conditions that are selective for lcII mutants (figure 2, path A). In addition, a dilution of infected G1250 cells is plated and grown under nonselective c
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