How were the yeast knockout strains verified?
Deletion Strain Confirmation:
The correct replacement of the gene with KanMX was verified in the mutants by the appearance of PCR products of the expected size using primers that span the left and right junctions of the deletion module within the genome. Four ORF-specific confirmation primers (A, B, C, and D primers) were chosen for each ORF disruption.
The "A" and "D" primers were positioned 200-400 bp from the start and stop codons of the gene, respectively. The "B" and "C" primers were located within the coding region of the ORF and, when used with the A or D primers, gave product sizes between 250-1000 bp. The "KanB" (5'-CTGCAGCGAGGAGCCGTAAT-3') and "KanC" (5'-TGATTTTGATGACGAGCGTAAT-3') primers are internal to the KanMX4 module.
For haploid or homozygous isolates, the junctions of the disruption were verified by amplification of genomic DNA using primers "A" and "KanB" and primers "KanC" and "D". Deletion of the ORF was verified by the absence of a PCR product using primers "A" with "B" and "C" with "D". In the case of heterozygous strains a successful deletion was indicated by the additional appearance of a wildtype-sized PCR product in reactions 3, 4 and 5. Finally, each deletion mutant was checked for a PCR product of the proper size using the primers flanking the gene.
In addition, each strain background was checked for the appropriate auxotrophic markers and mating capabilities.