Reece-Hoyes John S, Walhout Albertha J M
Cold Spring Harb Protoc. 2018 Jul 2;2018(7):2018/7/pdb.prot094979. doi: 10.1101/pdb.prot094979.
Generating DNA-binding domain (DB)-bait strains for Gateway-compatible yeast two-hybrid (Y2H) screens involves three steps. The first is to generate an Entry clone containing a DNA fragment encoding the protein of interest (e.g., an open reading frame, ORF). The second is to transfer this DNA fragment from the Entry clone to the Y2H Destination vector, pDEST32. The final step is to transform this construct into the Y2H yeast strain, MaV103. This protocol takes 24-37 d plus sequence confirmation, if necessary, to complete.
为Gateway兼容的酵母双杂交（Y2H）筛选生成DNA结合结构域（DB）诱饵菌株涉及三个步骤。第一步是生成一个入门克隆，其中包含编码目标蛋白的DNA片段（例如开放阅读框，ORF）。第二步是将该DNA片段从入门克隆转移到Y2H目的载体pDEST32中。最后一步是将该构建体转化到Y2H酵母菌株MaV103中。该方案需要24 - 37天，如有必要还需进行序列确认才能完成。
