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. 2005 Apr;4(4):733–741. doi: 10.1128/EC.4.4.733-741.2005

FIG. 3.

FIG. 3.

(A) MRS and lone RB2 deletion verification by PCR. PCR verification of the deletion of the MRS from chromosome 5 (lanes 3 to 10) and the lone RB2 from chromosome 3 (lanes 12 to 13). Lanes 2 and 11 were run with DNA from CAI4 and serve as a negative control. i. Lanes 2 to 10 show the PCR with primers 5 MRS HOK√ and URA3 DETECT F. Lanes 11 to 13 show the reaction with 3RB2 √ F and URA3 DETECT F. The presence of a 1.9-kb band (lanes 2 to 10) or a 1.7-kb band (11 to 13) indicates proper integration of the deletion cassette on one side. PCR was run with DNA from strains 5a MRSΔ-2 (lane 3), 5α MRSΔ-4 (lane 4), 5α MRSΔ-14 (lane 5), 5α MRSΔ-22 (lane 6), 5a MRSΔ-39 (lane 7), 5a MRSΔ-79 (lane 8), 5a MRSΔ/Δ-2 (lane 9), 5α MRSΔ/Δ-4 (lane 10), 3 RB2Δ-1 (lane 12), and 3 RB2Δ-2(lane 13). Lanes 1 and 14 contained size markers, and the sizes of relevant bands are shown at the right. ii. PCR with primers 5 MRS RB2√ (lanes 2 to 10), 3RB2 √ R (lanes 11 to 13), and URA3 DETECT R (lanes 2 to 13). The presence of a 1.0-kb (lanes 2 to 10) or a 1.2-kb (11 to 13) band indicates proper integration of the deletion cassette on the opposite side. iii. PCR with primers 5 MRS HOK√ and 5 MRS RB2√ (lanes 2 to 10) or 3RB2 √ F and 3RB2 √ R (11 to 14). The presence of a 2.2-kb band indicates proper integration of the deletion cassette on both sides. Due to the small size of the lone RB2 on chromosome 3, the flanking deletion detection primers also produce a 3.6-kb product across the intact lone RB2, and this bandshows up in both CAI4 and the three RB2 heterozygous deletion strains where one chromosome still contains an intact lone RB2. (B) MRS deletion verification by CHEF gel; CHEF gel of the chromosome 5 MRS deletion. Strains CAI4 (lanes 1 and 10), 5a MRSΔ-2 (lane 2), 5α MRSΔ-4 (lane 3), 5α MRSΔ-14 (lane 4), 5α MRSΔ-22 (lane 5), 5a MRSΔ-39 (lane 6), 5a MRSΔ-79 (lane 7), 5a MRSΔ/Δ-2 (lane 8), and 5α MRSΔ/Δ-4 (lane 9) are shown. In each of the heterozygous MRS deletion strains (lanes 2 to 7), one chromosome 5 homologue has decreased in size equivalent to the size of the deleted MRS. The homozygous MRS deletion strains (lanes 8 and 9) show the same decrease in both chromosome 5 homologues. Chromosomes 5, 6, and 7 are indicated on the right, with relevant sizes indicated on the left.

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