Figure 4

Recovery of the cbp1 mutant from mutant pool 12. (A) Diagram showing the addressing strategy used to efficiently identify which of 96 constituents of pool 12 correspond to the targeted cbp1 mutant. Individual clones were arrayed into 96-well plates and sub-pools created representing each row (letters) and column (numbers). Shaded wells depict the desired cbp1::T-DNA insertion clone or row and column sub-pools containing the clone. (B) Identification of the clone corresponding to the cbp1::T-DNA mutant. PCR was performed on each column and row sub-pool with the RB6 and CBP1-23 primers. Positive PCR amplicons identified the isolate at B4 as the cbp1::T-DNA mutant. (C) Southern blot analysis of the mutant strains with T-DNA insertions. Hind III-digested genomic DNAs prepared from OSU4, WU15, and OSU8 strains were probed with a T-DNA-specific probe. Single 3.8 kb and 3.0 kb bands detected in OSU4 and OSU8, respectively, indicate the mutant strains do not harbor multiple integrations of the T-DNA element.