Figure 5
PCR assay to screen for knockout alleles of mcm, pcn, pri1, and pri2 in Halobacterium sp. NRC-1. A. For the top four panels, lanes 1–20 contain product obtained from individual PCR reactions using total genomic DNA extracted from 20 individual Foar colonies as template and primers which reside ~500 bp 5' and 500 bp 3' to either mcm, pri1, and pri2 or ~1000 bp 5' and ~1000 bp 3' to pcn. For mcm, pri1, and pri2, predicted knockout alleles would be ~1000 bp in size, while wild-type alleles are approximately 2500, 1300, and 1100 bp larger, respectively. For pcn predicted knockout alleles would be ~2000 bp in size, while wild-type alleles are approximately 800 bp larger. For pri1 and pri2 NS refers to a nonspecific PCR based artifact that is observed when using those specific primer sets. B. The two panels at the bottom show the same screens as above, but after using Halobacterium sp. NRC-1 derivatives with a replicating plasmid containing a wild-type copy of the mcm or pri2 gene plus the entire 5' intergenic region. Either the ~1,000 bp knockout alleles or larger wild-type alleles are observed.