中药复方研究室细胞与分子实验操作指南（2009.9）2/32目录第一章细胞实验操作·········································································3第一节细胞培养常用耗材与设备·······················································3第二节无菌操作············································································3第三节培养用品的清洗···································································5第四节培养用品的消毒灭菌及灭菌锅的使用········································6第五节常用缓冲液、胰蛋白酶消化液及培养基的配制····························8第六节细胞培养、换液、消化传代···················································11第七节细胞计数···········································································14第八节细胞复苏与冻存··································································16第九节MTT法测定细胞活力或细胞增殖············································17第二章分子实验操作········································································17第一节PCR引物设计与实验操作······················································17第二节WESTERNBLOT实验操作·······················································26第三节FLOWCYTOMETRY实验操作····················································323/32第一章细胞实验操作第一节细胞培养常用耗材与设备1.准备室中常见耗材与设备去离子水制备器、酸缸、烘箱、高压灭菌锅、储备柜（放置干净未消毒的物品）。容量瓶（500ml，1L）、锥形瓶（500ml，1L）、磁力搅拌器、pH计、电子精密天平、台式离心机、冷冻离心机、涡旋器、普通冰箱（4℃，-20℃）、超低温冰箱（-70℃）。细胞培养瓶（25mm2，75mm2，175mm2）、细胞培养板（96-well，24-well，12-well，6-well）、细胞培养皿（35mm，60mm）、细胞冻存管（2ml，5ml）、离心管（10ml）、移液管（2ml，5ml，10ml）。移液枪（2.5μl，10μl，100μl，200μl，1000μl，5ml），枪头（10μl，200μl，1ml；颜色分别为：白，黄，蓝），eppendorf管（0.5ml、1.5ml）。2.细胞房中常见耗材与设备超净工作台、二氧化碳培养箱、恒温水浴锅、倒置显微镜、普通冰箱（4℃，-20℃）。酒精喷壶（装75%酒精），酒精棉（浓度为75%）、纯酒精（>95%，酒精灯使用）第二节无菌操作1.无菌室的打扫与灭菌1)定期打扫无菌室：先用自来水拖地、擦桌子、超净工作台等，然后用3‰新洁尔灭擦拭。2)培养箱定期灭菌：用75%酒精擦拭孵箱内部（最好同时将孵箱中各隔层拆下），再用紫外灯照射。（对于含有自动灭菌程序的孵箱，先用75%酒精擦4/32拭后，再启动其灭菌程序）3)实验前细胞房与超净台的灭菌：打开紫外灯，照射30min左右。4)每天实验结束后，应及时清理垃圾筒中垃圾。2.实验人员的无菌准备1)实验前应用肥皂洗手。2)穿好实验服，更换拖鞋。3)75%酒精消毒双手。3.操作台中的无菌操作1)在超净台经紫外照射灭菌...
