Sample ID:A(Luc48)B(Luc24off24)C(ER48) D(ER24off24)E(KR48) F(KR24off24)G(EV48) H(EV24off24)I(KV48) J(KV24off24) 預計流程(可再議)各細胞maintain者在星期五種三個十公分盤細胞在含藥培養其中,分別為Monday(2M),Tuesday(1.5M), Wednesday(1M)。隔週週一,取Monday盤細胞,種兩個6孔盤(0.5M/well)培養在不含藥的tet-free medium中。週二加dox,週三取一盤6孔盤做wash-off步驟,週四收細胞(五格for RNA,一格for protein)。這是三個重複中的第一組。三個重複中的第二組:週二取Tuesday盤細胞,種兩個6孔盤(0.5M/well)培養在不含藥的tet-free medium中。週三加dox,週四取一盤6孔盤做wash-off步驟,週五收細胞(五格for RNA,一格for protein)。三個重複中的第三組:週三取Tuesday盤細胞,種兩個6孔盤(0.5M/well)培養在不含藥的tet-free medium中。週四加dox,週五取一盤6孔盤做wash-off步驟,週六收細胞(五格for RNA,一格for protein)。哪 一組細胞送抽RNA做microarray呢?把30個protein sample先抽好做flag及p21測試,看induction及wash-off情形再定。因為要抽RNA,所以所有細胞harvest下來後均勻拍 散,全部先放進-80C冰箱待用。5M細胞約可抽出50ug RNA,而我們的需要量是3~5ug左右。——————————————————————————————–2008/10/16 SFL wrote:Purpose: To analyze transcriptome changes modulated by R. Fact1: Treatment of TetER for 24h, remove dox, ER is depleted in 24h.Fact2: Treatment of TetKR for 24h, remove dox, KR is depleted in 24h.Fact3: