, Add 2 g of amino acid mixture prepared as follows: 2 g Adenine hemisulfate, Complete Synthetic Drop Out (SD): dissolve 26.7 g of minimal SD base with glucose in 1 L of water

, SD containing every essential amino acids except those specified (-L:-Leucine ;-W:-Tryptophan, Histidine)

, YPGLU: For 1 L, weight 10 g of yeast extract, 10 g of Bacto Peptone and 20 g of glucose. Complete with 25 g Bacto agar for solid media

, YCM: Same as YPGLU

, Use frozen aliquots of pre-transformed Y187 yeast strain (mating type ?) containing a cDNA library cloned in pACT2, Check cell viability by plating serial dilutions on SD-L plates

, Clone the pathogen ORF into pGBKT7 vector, and transform the recombinant plasmid using a standard procedure into AH109 yeast strain (mating type a)

, Incubate at least two days 30 °C in a humidified incubator. Plates can be stored at 4 °C until the two-hybrid screening, Spread pGBKT7-transformed AH109 yeast on SD-W plates

, ), a competitive inhibitor of the HIS3 enzyme, to counteract the potential self-transactivation of the HIS3 reporter gene by the pathogens' proteins. Perform an auto-activation test by determining the concentration of 3-AT, which prevents growth of pGBKT7

, Seed 30 ml of SD-W with few colonies of pGBKT7-transformed AH109. Incubate 30 hr at 30 °C with rotation

, Seed 200 ml of SD-W with the 30 ml of the AH109 cultures. Incubate with rotation around 20 hr at 30 °C

, Incubate thawed cDNA library-transformed Y187 in 20 ml YPGLU, incubate 10 min at 30 °C with rotation

, Centrifuge a volume of pGBKT7-transformed AH109 culture corresponding to an equivalent amount of viable Y187 yeast cells for 5 min at 3,500 rpm at 20 °C. Carefully withdraw the supernatant and resuspend pellet in 10 ml YPGLU

, Transfer in a 50 ml tube. Centrifuge for 5 min at 3,500 rpm at 20 °C, carefully withdraw supernatant

, Spread yeast on to 3 YCM-Agar 150 mm plates, 0.5 ml/plate. Incubate 4.5 hr at 30 °C

, Collect mated yeast from YCM plates by scraping with a rake glass in 10 ml of SD-L/-W/-H. Rinse the plates two times with 5 ml SD-L/W/-H medium and pool

, Centrifuge 5 min at 3,500 rpm, 20 °C. Discard supernatant and resuspend the yeast pellet in 5 ml SD-L/-W/-H medium

, 5 ml/plate) of SD-L/-W/-H Agar supplemented with the appropriate concentration of 3aminotriazole (3-AT) determined in the auto-activation test. Incubate at 30 °C in a, p.10

, Determine the diploid (2n) rate in order to evaluate mating efficiency by spreading 250 ?l of serial dilutions (10-4 to 10 on SD-L/-W plates. Incubate plates at 30 °C for two days. Count colonies grown on SD-L/-W, and deduce the total diploid number present in the initial volume of mated yeast

, 6-10 days after incubation at 30 °C, pick mated yeast colonies in fresh SD-L/-W/-H + 3-AT plates. [Tip: order the yeast in a scheme of 8 lanes of 12 wells reproducing a 96 well plate so that it will be easier afterward for sequencing

, Screening of his3 positive clones by PCR and sequencing The goal is to PCR amplify the ORF contained in the pACT2 vectors of yeast colonies grown on selective SD-L/-H/-W plates. 1. Put a 96 well PCR plate on ice. 2. To lyse yeast cells, add 50 ?l per well of a Zymolase 20T solution (2.5 mg/ml in 10 mM HEPES

, Gently resuspend one colony per well

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