Verification of the ΔlasR, ΔkynBU, trpE-phnAB-ΔkynBU and ΔHSI-II mutants by PCR. A. Verification of single gene deletion. To confirm that the target gene is mutated, two primer pairs Fupout/Rin-kan and Fupout/Rdownout were used. The ΔlasR mutant was verified with primer pairs Fupout-lasR/Rin-kan and Fupout-lasR/Rdownout-lasR, ΔkynBU and trpE-phnAB-ΔkynBU with Fupout-kynBU/Rin-kan and Fupout-kynBU/Rdownout-kynBU. B) Verification of deletion of large chromosomal region. To confirm that the MAR2xT7 transposon replaced the 24 kb HSI-II locus, primer pairs Fdown-MAR2xT7/Rdown-PA14_42880 and Fup-PA14_43100/Rdown PA14_42880 were used. No amplification is achieved when wild type genomic DNA is used.
Lesic and Rahme BMC Molecular Biology 2008 9:20 doi:10.1186/1471-2199-9-20