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Growing colonies were identified after 24 h of 37 °C aerobic conditions incubation using mass spectrometry with MALDI™ Biotyper system (Bruker Daltonics, Germany).
The suspensions (OD600, 0.1) for the second EB treatment were prepared by growing colonies on KB medium at 24 °C for 24 48 h.
Growing colonies were selected and plasmid DNA was recovered from E. coli.
Recombinant pACT2 plasmids of growing colonies were isolated and subsequently transformed in E. coli for plasmid amplification and isolation.
Survival rate (triplicate/sample) was calculated as a ratio of the number of growing colonies to the number of spores inoculated.
Fast growing colonies were subsequently picked from these –Arg plates and streaked onto –Ura plates to assay alleviation of silencing at outer repeats (otr).
Our data show that digital imaging of cellular autofluorescence detects growing colonies much earlier than the traditional visual plate counting method.
Most growing colonies will be correct.
The double mutants formed small, slower growing colonies.
Approximately 15 of these slower-growing colonies were restreaked and stored following the same protocol as for the faster growing colonies.
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