Deletion mutants were confirmed by colony PCR with appropriate primers sequencing of the PCR product to check the presence of unique bar codes.

We verified the mutant using colony PCR method: PCR enzymes directly reach the chromosomal DNA after breaking the cell wall of mutant colony by enzyme or physical forces. To know whether the mutant grown in G418 plate is the final deletion mutant of target gene or not, we prepared two kinds of gene-specific primers for 5' upstream and 3' downstream of target gene. One, named CP5 was used for colony PCR with common CPN1 and CPN10 primers at the N terminus and the other, named CP3, is for CPC1 and CPC3 primers at the C terminus of the KanMX module, respectively. Finally, we were able to confirm the mutants based on the size of the PCR fragments.

To confirm deletion mutant for a target ORF, the tag sequence was amplified by colony PCR using a primer specific to the target gene and the common primer in KanMX module.

Sequences of CPN1, CPN10, CPC1, CPC3 in KanMX module
CPN1    5-CGTCTGTGAGGGGAGCGTTT-3
CPN10  5-GATGTGAGAACTGTATCCTAGCAAG-3
CPC1    5-TGATTTTGATGACGAGCGTAAT-3
CPC3    5-GGCTGGCCTGTTGAACAAGTCTGGA-3