细胞结合实验.docxVIP

细胞结合实验 细胞结合实验 1| Prepare cells for the experiment. This procedure will present the detailed cell-SELEX protocol for both suspension and adherent cultured cell lines. For adherent cells, there are two options: the experiment can be performed either directly in a culture dish or using dissociated cells. If using dissociated cells, prepare them by following the steps given in Box 1 before following the procedure given below. If performing the experiment in a culture dish, refer to Box 2 and continue with the procedure from Step 61. Initial Dna library pool preparation ● tIMInG 20 min 准备实验细胞。这个程序将详细的细胞SELEX协议和悬浮 贴壁培养细胞系。贴壁细胞，有两种选择：实验可以直接在 或使用解离的细胞培养皿。如果使用解离的细胞，他们准备通过下面1盒之前的步骤 下面给出的程序如下。如果在培养皿中进行实验，涉及2盒并继续 步骤61步骤。最初DNA文库池制备●定时20分钟 2| Add 20 μl of 0.5 mM (10 nmol) DNA library to 350 μl of binding buffer, mix and heat the mixture at 95 °C for 5 min. Snap-cool on ice and keep on ice until ready to use (same day). pause poInt If selection is not accomplished on the same day, store the DNA library pool at ? 20 °C. Thaw library on ice whenever ready to use. The denaturation step is not necessary once DNA has been thawed on ice. crItIcal step Heating the DNA at 95 °C and subsequent fast cooling on ice are important to create folded ssDNA. preparation of target cells: cell viability ● tIMInG 30 min 添加20μL 0.5毫米（10 nmol）350μL的结合缓冲液DNA文库，混合和热的混合物在95°C 5分钟。 卡酷冰保持在冰上待用（同一天）。 暂停点如果选择不在同一天完成，在?20°C.解冻库在冰店DNA文库池 当准备使用。变性步骤是不必要的一旦DNA已解冻的冰。 关键步骤中加热到95°C和随后的快速冷却的冰上的DNA是创造折叠ssDNA重要。 靶细胞的制备：细胞活力●定时30分钟 3| Determine cell viability using Trypan blue exclusion assay. crItIcal step Cell viability assessment is very important, especially for suspension cells. Too many dead cells will seri- ously affect the efficiency of selection. DNA can nonspecifically adhere to and enter dead cells. This will cause the loss of important sequences, delay of enrichment or even the failure of selection. As the highest cell viability is ideal, it is preferable to use more than 95% viable cells. preparation of ta