The CAN1 fluctuation analysis was performed as described in57 (link). Relevant genotypes for the CanR mutation assay were streaked out 48 h prior inoculation to conserve population doublings within replicates. At least 14 independent single colonies from each genotype were entirely excised from the agar plate using a sterile scalpel to inoculate a 10 ml of YPD medium. The cultures were incubated at 30 °C, 250 rpm for 16 h. After measuring the optical density of the cultures they were harvested by centrifugation. Then, each culture was resuspended in 1 ml sterile water. Exactly 1 ml of each resuspension was transferred to a new tube. From this, a 10-fold dilution series up to a dilution factor of 106 was performed in a 96-well plate. Finally, 100 µl of all strains from the 10−6 dilution were plated on a YPD plate and distributed with exactly four glass beads per plate. All strains were plated on SC-ARG plates supplemented with 60 µg/mL canavanine with the indicated dilution factor. The plates were incubated for 72 h at 30 °C before the outgrown colonies were manually counted. The medium for the CanR mutation assay was mixed, autoclaved and poured each day before plating to maintain constant conditions between replicates.
For evaluation, the number (#) of mutant cells per culture, representing r, was calculated: r=culturevol.×conc.factor×#mutantcoloniesplatedvol.×dilutionfactorplated
The following correction was used to account for the progenies of each individual CAN1 mutation event per cell. With M being a scaled value that represents the number of cells that have actually undergone a mutation event (from which the counted progenies originated): r=M(1.24+lnM)
The final mutation rate was calculated dividing M by the total number of cells present in the initial culture: mutationrate=M#cellsintheculture
The data was plotted as the Median with 95% Confidence interval using the GraphPad PRISM8 software.
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