, 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
Prepare the following mix: 525 ?l 10X ExTaq buffer, 420 ?l dNTPs mix (2.5 mM), 10.5 ?l Forward Primer (1 ?g/?l), 10.5 ?l Reverse Primer (1 ?g/?l), 31.5 ?l ExTaq (5 U/?l) and 3202.5 ?l H 2 0. Distribute 40 ?l of the mix per well in a 96 well PCR plate. Add 10 ?l of lysed yeast per well. Perform amplification as follows: 94 °C for 3 min, then 35 cycles at 94 °C for 30 sec, 60 °C for 1 min, 72 °C for 4 min, and end with 72 °C for 7 min, Incubate 15 min at 37 °C and 15 min at 95 °C. 5. Put the plate back on ice, vol.1, pp.159-169, 2011. ,
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