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Automated Protocol for Protein Interaction Selection in Yeast

This detailed protocol outlines a robotizable double-hybrid transformation and selection method based on p53 and largeT protein interactions. The procedure involves specific dilutions and growth assessment in a deep-well plate setup.

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Automated Protocol for Protein Interaction Selection in Yeast

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  1. Transformação 2 Transformação 1 pISCA (1g) pSEMPRESA (1g) pPRESA (0ng) pISCA (1g) pSEMPRESA (1g) pPRESA (100ng) 1:100 1:20 1:10 SDs sem histidina 3AT 0 ou 2,5mM 1 ng 0 ng 5 ng 10 ng pISCA + pSEMPRESA pPRESA 0 ng 1 ng 5 ng 10 ng 22 setores controle 4 placas grandes = 1 placa deepwell Figura 9: Esquema do protocolo de transformação e seleção de duplo-híbrido robotizável baseadas na interação entre as proteínas p53 e largeT. De duas transformações, cada uma contendo 1 mg de pISCA (Gal4DL-p53), 1 mg de pSEMPRESA (Gal4DA) e 0 ou 100ng de pPRESA (Gal4DA-largeT), foram tomadas alíquotas correspondentes a 0; 1; 5 e 10ng de pPRESA em tubos de cultura SDs líquida sem histidina suplementado com 3AT 0 ou 2,5mM e distribuídos em 96 poços de placa deepwell(400L por poço). O crescimento foi avaliado pela medida de densidade do meio líquido em espectrofotômetro com leitor de placa de ELISA (OD600nm) segundo protocolo de diluições F (Material e Métodos, item 3.15).

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