Figure 5.—

Verification analysis. (A) DNA blot verification. Genomic DNA was isolated from plants carrying bti00245::Ac (left, lanes 1 and 2) or bti00194::Ac (right, lanes 1 and 2) and from progenitor Ac-containing P1-vv (lanes 3) and the Ds reporter plants (lanes 4). Lane 5 contains DIG-labeled DNA VII ladder (Roche). DNA was digested with EcoRI and fractionated on 0.8% agarose gels. DNA blot analysis was performed with a DIG-labeled flanking sequence probe of 443 bp (bti00245::Ac) or 432 bp (bti00194::Ac) generated using the Ac-end primer and flanking sequence-specific primer pairs [DIG-labeled VII ladder (lane 5)]. (B) PCR verification for the presence of bti00245::Ac (left) and bti00194::Ac (right). Verification PCR was performed using DNA from plants that carried the tr-Ac element (lanes 1 and 2), the progenitor P1-vv allele (lane 3), and the r1-sc:m3 allele (lane 4). An Ac-end primer was used with a gene-specific primer to amplify fragments of 443 and 432 bp from bti00245::Ac and bti00194::Ac, respectively. Lanes 5 and 6 represent positive (plasmid template) and negative (no DNA) controls; lane 7 contains a 1-kb DNA ladder (Roche).