Restriction fragment analysis of BAC clones and genomic DNA. Selected 22-kD α zein-positive BAC clones and BSSS53 genomic DNA were digested with HindIII and separated by a 1% agarose gel. After transfer to a nylon membrane, the DNA was sequentially hybridized with the following: (1) a mixture of php200725 and ORF3 (found in the 3′ region of the cluster); (2) azs22;13 (most diverged 22-kD α zein); (3) LKR (lysine-ketoglutarate reductase)-like gene (not present on any BAC clone, as a negative control), and (4) a mixture of six different 22kD α zein cDNAs of BSSS53. Use of different probes allowed us to see which BAC clone(s) contains which gene(s). Moreover, higher hybridization stringency can be applied to eliminate cross-hybridization with 19-kD α zein genes. After each hybridization experiment, the membrane was exposed and a picture taken to record each additional positive band, which results in a composite figure. Because of the different amounts of genomic DNA (∼10 μg) and BAC DNA (∼50 ng each) loaded on the gel, the genomic DNA migrated more rapidly than the BAC samples. Samples from left toright are as follows: a size marker, BSSS53 genomic DNA, BAC 134, BAC 218, BAC 171, BAC 204, BAC 124, BAC 55, BAC 158, and BAC 193. BACs in lanes 8–10 contain the single α zein geneazs22;16, which can also serve as a copy number control for the other clones. Note that the lanes are slightly shifted due to overloading genomic DNA so that smaller restriction fragments can be detected.