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Y1H assay

AV Arun Kumaran Anguraj Vadivel
TM Tim McDowell
JR Justin B. Renaud
SD Sangeeta Dhaubhadel
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Y1H assays were carried out by following the Matchmaker® Gold Yeast One-Hybrid Library Screening System User Manual (Clontech, USA). The recombinant plasmid p30bpTR-AbAi was transformed into yeast (Y1H Gold strain) using the Yeastmaker™ Yeast Transformation System 2 (Clontech, USA) and grown on SD/-Ura media at 30 °C for 3 days. The transformed colonies were screened by colony PCR using Matchmaker Insert Check PCR Mix 1 (Clontech, USA). Y1H assays were performed by following the Matchmaker Y1H user manual (Clontech, USA). The minimum inhibitory concentration of aeurobasidin A (AbAi) for yeasts carrying 30bpTR promoter bait was 150 ng/mL. The prey constructs (pGADT7-GOI) were transformed into yeast carrying a promoter bait fragment, plated on SD/-Leu/AbAi150, and incubated at 30 °C for 3–5 days.

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