Figure 4
PCR assay to screen for knockout alleles of DNA polymerase genes polD1, polD2, polB1, and polB2 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 5-Foar colonies as template and primers which reside ~500 bp 5' and 500 bp 3' of the specific ORF targeted for deletion. For polD1, polD2, polB1, and polB2, knockout alleles are ~1000 bp in size where obtained, while wild type alleles are approximately 1200, 4100, 2700, and 2200 bp larger, respectively. B. The two panels at the bottom show the same screens as above, but using Halobacterium sp. NRC-1 derivatives with a replicating plasmid containing a wild-type copy of the polD1 or polB1 gene plus the entire 5' intergenic region. Only the ~1,000 bp knockout alleles are observed.