Propyzamide reference material

Propyzamide Reference Material, Oryzalin Standard, and colchicine were obtained from Fujifilm Wako Pure Chemical Co., Japan. The strains tub2-A, tua1-A x tub1-B (double disruptant) or tua2-A x tub1-B (double disruptant) were grown to the mid-log phase and then irradiated by ultraviolet light until about 50% of the cells were killed. The culture was spread on TAP/agar plates containing 20 μM propyzamide or 10 μM oryzalin, kept in the dark for 12 h, and then incubated under light for 5–10 days. Colonies that appeared were transferred to liquid medium in 96-well plates containing the same concentration of propyzamide or oryzalin. From each culture that grew, genomic DNA was extracted and subjected to PCR using the following primers: 5'-ChlaTuA1_long969 and 3'-TuA6260 (for tua1), 5'-Tua2-10g and 3'-TuA2-3288g (for tua2), 5'-tub1-33c and 3'-tub1-1667c (for tub1), and 5'-EcoTuB2-upper and 3'-XhoTuB2-lower (for tub2). The PCR products were processed for DNA sequencing (Macrogen Japan Co.).