Genomic DNAs were obtained from vegetative cells grown at 37°C on 5% horse blood agar plates. DNA was purified using the QIAGEN® Genomic-tip 100/G columns and QIAGEN® Genomic DNA Buffer Set. Briefly, bacterial colonies were harvested by scraping the agar surfaces from 16 to 18 h-old Petri dishes. Cell pellet was resuspended in 3.3 ml of Buffer B1 containing 7 μl of RNase A (100 mg/ml, QIAGEN) and incubated at 37°C for 45 min with 350 μl of lysozyme (100 mg/ml, Roche) and 100 μl of proteinase K stock solution (QIAGEN). Following addition of 1.2 ml of Buffer B2, DNA lysate was further incubated at 50°C for 1 h. Particle-free sample was next applied to the equilibrated QIAGEN Genomic-tip and DNA purification on anion-exchange resin processed according to the manufacturer’s recommendations. After isopropanol precipitation, genomic DNA was resuspended in 400 μl of 10 mM Tris HCl (pH 8) for at least 2 h at 50°C.
DNA solutions were transferred to a 0.22 μm sterile Ultrafree-MC spin filter (Millipore) and centrifuged for 2 min at a maximal speed of 8000 × g to ensure the complete removal of live forms of B. anthracis from DNA. Viability testing was systematically performed before DNA was taken out of the BSL-3 facility. An aliquot of each DNA preparation (a quarter) was spread on Petri dishes and grown at 37°C for 24 h.
DNA solutions were transferred to a 0.22 μm sterile Ultrafree-MC spin filter (Millipore) and centrifuged for 2 min at a maximal speed of 8000 × g to ensure the complete removal of live forms of B. anthracis from DNA. Viability testing was systematically performed before DNA was taken out of the BSL-3 facility. An aliquot of each DNA preparation (a quarter) was spread on Petri dishes and grown at 37°C for 24 h.