Methods for identifying gene families in the minimum tile path of BAC clones. Panel A): The strategy for probe pool designs. Panel B): An example of three positive clones from colony hybridizations to double spotted colonies from four 384 plates per location. Panel C): DNA from BAC clones identified from the MTP hybridizations were restriction digested with the restriction enzyme used for insertion, transferred to a membrane and hybridized to the EST probe. (a): The BAC clones B48B23, H59N09, and H76L07 (Lane 1, 2, and 3) were positive for the identified EST FiS1H9 (BI273631 a histone H2A orthologue) from the initial MTP hybridization. (b): DNA of BAC clones B23A05, B23C13, B38M08, B48M07, H15A05, H15A06, H20G14, H36D08, and H42K03 (Lane 1, 2, 3, 4, 5, 6, 7, 8, and 9 respectively) were hybridized to the EST Fi36H18 (BI347330 a translational elongation factor 1B-alpha 1 orthologue) identified from the initial MTP hybridization for second confirmation. The hybridization proved that 6 out of the 9 (Lane 2, 3, 4, 5, 7, and 8) initial positives were positive with a second Southern hybridization.