ERα 接受器轉植至 Raw264.7 細胞相關實驗

材料:

Medium、1% Trypsine(GIBCO)、細胞培養基(dish)、無菌 1×PBS、

1000p 吸管唧筒(pipette pump)、電動吸量管(Electric Pipette)、

無菌操作檯、培養箱(37℃、5% CO²)。

方法:

Raw264.7 細 胞 株 ， 以 含 有 10% 胎 牛 血 清 (Fetal Bovine serum;GIBCO) 、 1% Peniciline-streptomycin (GIBCO) 、 1%

L-Glutamine(GIBCO) 、 1ml Fungizone Amphotericn B (GIBCO) 的 RPIM(GIBCO)培養液將其培養在 5%二氧化碳、恆溫 37℃的培養箱中，

每三天更換一次培養液；當細胞生長至九分滿，及以 1% Tryosine 將 細胞游離下來，移至新培養基中重新貼附生長。

1-2 質體翠取 材料:

QIAprep Spin Miniprep Kit、1000p 吸管唧筒(pipette pump)、

微量離心管(eppendorff)、離心機。

方法:

挑菌養在 3ml 含有 Ampicillin 的 LB medium 中培養 20 小時，利 用 QIAprep Spin Miniprep Kit 翠取質體 DNA。首先將菌液離心 (3000rpm、4℃、5 分鐘)倒掉上層液，加 250ul Buffer P1 回溶、打 散沉澱下的菌體，將其移至 eppendorf ，加 250ul Buffer P2 上下 顛倒輕輕搖動於室溫作用 5 分鐘，在加入 350ul buffer N3 輕輕搖動，

讓豆花狀的 Genomic DNA 結聚，冰上作用 5 分鐘，以 12000rpm、4℃

離心 10 分鐘，取上層液到 QIAprep column，離心 30~60sec 後倒掉 離下來的液體，再加 500ul buffer PB 沖洗 QIAprep spin column，

離心 50~60sec，倒掉液體，再加 750ul buffer PE 沖洗 QIAprep spin column，離心 30~60sec 後倒掉液體，最後空的 column 再離心 1 分鐘 排除所有殘留液體，將 QIAprep column 移至於 1.5ml eppendorf，

接著加 50 ul buffer EB 或 ddH2O 到 QIAprep column 中心停滯 1 分 鐘，1400rpm 離心 1 分鐘，離心下來的液體即含有質體 DNA。

1-3 DNA 轉植(Transfection) 材料:

Raw264.7 細胞、無 FBS 及抗生素的 RPMI medium、只有 FBS 的 RPMI medium、Invitrogen Lipofectamine 2000 transgection kit

(GIBCO)、ER-α DNA、1000p 及 20p 吸管唧筒(pipette pump)、微量 離心管(eppendorff)、

步驟:

1×10⁶細胞天於 60cm dish，將其 mediumc 換成無 FBS 及抗生素的 RPMI medium，選用 Invitrogen Lipofectamine 2000 transgection kit，利用 Lipofection 的原理進行基因轉質的實驗。首先將質體 DNA 與適量無 FBS 及抗生素的 RPMI medium 混合，再依外送 DNA 量，抽取 1:1 比例的 Lipofectamine 與無 FBS 及抗生素的 RPMI medium 混合，

室溫作用 5 分鐘。接著將兩者以 Tip 混合 100 次，置於是溫 20 分鐘 後加入含有細胞的 dish 中，放置培養箱養 6 小時，之後將 medium 換 成只有 FBS 的 RPMI medium，置培養箱養 20 小時等其 ER-α 蛋白質 表顯。

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