Fig. 8. Complementation test procedure for S. cerevisiae. Mutants m1 through m4, in a and alpha cells with complementary nutritional markers, are streaked in parallel streaks across two separate complete plates. After the streaks have matured, they are replicated onto velveteen at right angles to each other. The streaks on the velveteen are then replicated onto a YPD plate and incubated at 30 degrees for 24 hrs to allow mating at intersections. The YPD plate is then replicated onto a plate with minimal medium (SD), and incubation at 30 degrees is continued for several more days. Only diploid cells--resulting from mating of the a and alpha cells at the intersections of the streaks and thus containing both the LEU2 and URA3 genes--can grow on the SD plate. The resulting diploid colonies are then tested for phenotype. Those colonies displaying the mutant phenotype (for example, the colony in the upper left, because it is homozygous for m1) have mutant genes in the same complementation group. Colonies displaying wild type phenotypes have mutant genes in different complementation groups.