分子生物学实验ppt课件

1、酶切PCR扩增，酶切载体片段 目的基因片段+衔接重组质粒转化重组子阳性鉴定NcoI CCATGG GGTACCXhoI CTCGAG GAGCTC37 ，OD600 1.0 加氯霉素 100 g/ml 37，过夜1.4 ml培育液4, 6000 rpm, 2 min沉淀加100 l 冰预冷溶液,猛烈振荡弃去上清，尽能够除净培育液室温，20 min200 l 新配制溶液，快速颠倒混匀冰浴5 min150 l 冰预冷溶液，温暖振荡冰浴5 min上清参与450 l饱和酚，混匀 4, 12,000 rpm，2 min上清参与450 l氯仿-异戊醇，混匀 4, 12,000 rpm，2 min上清参与3

2、00 l异丙醇，-20，30 min4, 12,000 rpm离心5 min500 l 70% 乙醇洗涤沉淀 4, 12,000 rpm，2 min弃上清，空气枯燥10 min20 l TE溶解质粒DNA4, 12,000 rpm，5 min（一）选择合适的水平电泳仪（二）选择孔径大小合适的点样梳（三）制备琼脂糖凝胶（四）用电泳缓冲液溶解凝胶，加入EB达0.5微克/毫升，摇匀，铺胶（五）在电泳槽中加入电泳缓冲液（六）待测的样品中加入指示剂，进行点样（七）打开电源开关，电压控制在5V/cm左右（八）待DNA各条带分开后，电泳结束l衔接反响体系 lddH2O 8.5l l10buffer 4.5l

3、l酶切DNA pBR322 20l l酶切DNA pUC18 10llT4 DNA ligase 1l l16 ，15 hl取10l衔接产物，核酸电泳上步剩余酶切产物做对照l 其他衔接产物用于转化37 ，190 rpmOD600 0.375，冰浴10 min4, 6000 rpm, 10 min弃上清，加4 ml冰预冷 0.1M CaCl2冰浴10 min， 4, 6000 rpm, 10 min弃上清, 参与1 ml 0.1M CaCl2，分装200 l/管 二转化反响分别参与衔接产物20 l, 阳性对照质粒pBR322 1 l, 阴性对照pUC18 1 l, 空白对照冰浴30 min42

4、, 90 s冰浴1-2 min弃500 l 上清, 衔接产物取100 l , 10 l各涂一平板, 空白，阳性对照和阴性对照200 l各涂一平板参与800 l LB培育基37 水浴5 min37 ，缓慢摇，复苏45 min37 ，12-16 h察看转化子的出现6000 rpm, 5 min2. 转化反应前一天，先倒皿铺好平板3. 取以上各组反应物在平板上涂布4.培养皿置于37培养箱中过夜5. 观察转化子出现的情况（三）倒皿铺平板1. 按照各组转化反应的混合物，控制不同的选择性培养基LB固体培养基+Ap（100ug/ml）+Tet（40ug/ml）供连接混合物转化，pUC 18转化用LB固体+A

5、p（100ug/ml）+X-gal（100ug/ml）+IPTG（119ug/ml）供连接混合物转化，pUC 18转化用共10皿（一）制备0.8%琼脂糖凝胶（二）取Eppendorf管，每管加50微升Cracking gel缓冲液（三）用扁头牙签，将菌落刮到Eppendorf管中（四）在37水浴中保温15分钟（五）取出各管，4，1,500r/min，离心15分钟（六）立即吸出各管上清液25-30微升，进行点样电泳（七）当溴酚兰迁移到凝胶板的2/3处时，停止电泳（八）将电泳凝胶染色30分钟（九）观察质粒的迁移距离，并拍照供进一步分析（一）在PCR反应管中加入2 PCR Premix 12.5ul

6、（二）向此反应管中加入 2ul Control Template 和10.5 ul的Primer Pair 1，混匀（三）向反应管中滴加一滴石蜡油（四）按以下条件进行PCR反应： 94 1分钟 1次 94 30秒钟 55 30秒钟 循环30次 72 30秒钟（五）取8ul PCR扩增液，用2%琼脂糖凝胶进行电泳4C1z)w&s!pYmUjRgOcL9I6E3B+y(v%r#oXlTiQeNbK8G5D2A-x*t$qZnVkShPdMaI7F4C0z)w&s!pXmUjRfOcL9H6E2B+y(u%r#oWlThQeNbJ8G5D1A-w*t$qYnVkSgPdMaI7F3C

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11、H6E2B+x(u%rZoWlThQeMbJ8G4D1A-w*t!qYnVjSgPdLaI7F3C0y)v&s#pXmUiRfNcK9H5E2B+x(u$rZoWkThQeMbJ7G4D1z-w*t!qYmVjSgOdLaI6F3B0y)v%s#pXlUiQfNcK8H5E2A+x(u$rZnWkThPeMbJ7G4C1z-w&t!qYmVjRgOdL9I6F3B0y(v%s#oXlUiQfNbK8H5D2A+A+x*u$qZnWkShPeMaJ7F4C1z)w&t!pYmUjRgOcL9I6E3B+y(v%r#oXlTiQfNbK8G5D2A-x*u$qZnVkShP

12、dMaJ7F4C0z)w&s!pYmUjRfOcL9H6E3B+y(u%r#oWlTiQeNbJ8G5D1A-x*t$qYnVkSgPdMaI7F4C0z)v&s!pXmUjRfOcK9H6E2B+y(u%rZoWlThQeNbJ8G4D1A-w*t$qYnVjSgPdLaI7F3C0y)v&s#pXmUiRfNcK9H5E2B+x(u$rZoWkThQeMbJ8G4D1z-w*t!qYnVjSgOdLaI6F3C0y)v%s#pXlUiRfNcK8H5E2A+x(u$u$rZnWkThPeMbJ7G4C1z-w&t!qYmVjRgOdL9I6F3B0y(v%s

13、#oXlUiQfNbK8H5D2A+x*u$rZnWkShPeMaJ7G4C1z)w&t!pYmVjRgOcL9I6E3B0y(v%r#oXlTiQfNbK8G5D2A-x*u$qZnVkShPdMaJ7F4C0z)w&s!p!pYmUjRfOcL9H6E3B+y(v%r#oWlTiQeNbK8G5D1A-x*t$qZnVkSgPdMaI7F4C0z)v&s!pXmUjRfOcK9H6E2B+y(u%rZoWlThQeNbJ8G4D1A-w*t$qYnVjSgPdLaI7F3C0z)v&s#pXmUiRfOcK9H5E2B+x(u%rZoWkThQeMbJ8G4

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15、pXmUjRfOcK9H6E2B+y(u%rZoWlThQeNbJ8G4D1A-w*t$qYnVjSgPdLaI7F3C0z)v&s#pXmUiRfOcK9H5E2B+x(u%rZoWkThQeMbJ8G4D1z-w*t!qYnVjSgOdLaI6F3C0y)v%s#pXlUiRfNcK8H5E2A+x(u$rZoWkThPeMbJ7G4D1z-z-w&t!qYmVjSgOdL9I6F3B0y)v%s#oXlUiQfNcK8H5D2A+x*u$rZnWkShPeMaJ7G4C1z)w&t!pYmVjRgOcL9I6E3B0y(v%r#oXlTiQfNbK8H5D2A-x

16、*u$qZnWkShPdMaJ7F4C1z)w&s!pYmUjRgOcL9H6E3B+y(v%r#oWlTiQeNbK8G5D1A-x*t$qZnVkSgPdMaI7F4C0z)w&s!pXmUjRfOcL9H6E2B+y(u%r#oWlThQeNbJ8G5D1A-w*t$qYnVkSgPdLaI7F3C0z)v&s#pXmUiRfOcK9H5E2B+x(u%rZoWlThQeMbJ8G4D1A-w*t!qYnVjSgPdLaI6F3C0y)v&s#pXlUiRfNcK9K9H5E2A+x(u$rZoWkThPeMbJ7G4D1z-w&t!qYmVjSg

17、OdL9I6F3B0y)v%s#oXlUiQfNcK8H5E2A+x*u$rZnWkThPeMaJ7G4C1z-w&t!pYmVjRgOdL9I6E3B0y(v%s#oXlTiQfNbK8H5D2A-x*u$qZnWkShPdMaJ7F4C1z)w&s!pYmUjRgOcL9I6E3B+y(v%r#oXlTiQeNbK8G5D2A-x*t$qZnVkShPdMaI7F4C0z)w&s!pXmUjRfOcL9H6E2B+y(u%r#oWlThQeNbJ8G5D1A-x*t$qYnVkSgPdMaI7F3C0z)v&s!pXmUiRfOcK9H9H6E2B+x(u%

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19、4C0z)w&s!pXmUjRfOcL9H6E2B+y(u%r#oWlThQeNbJ8G5D1A-x*t$qYnVkSgPdMaI7F3C0z)v&s!pXmUiRfOcK9H6E2B+x(u%rZoWlThQeMbJ8G4D1A-w*t!qYqYnVjSgPdLaI6F3C0y)v&s#pXlUiRfNcK9H5E2A+x(u$rZoWkThQeMbJ7G4D1z-w*t!qYmVjSgOdLaI6F3B0y)v%s#pXlUiQfNcK8H5E2A+x*u$rZnWkThPeMaJ7G4C1z-w&t!pYmVjRgOdL9I6F3B0y(v%s#oXlUi

20、QfNbK8H5D2A+x*u$qZnWkShPeMaJ7F4C1z)w&t!pYmUjRgOcL9I6E3B+y(v%r#oXlTiQeNbK8G5D2A-x*u$qZnVkShPdMaJ7F4C0z)w&s!pYmUjRfOcL9H6E3B+y(u%r#y(u%r#oWlTiQeNbJ8G5D1A-x*t$qYnVkSgPdMaI7F3C0z)v&s!pXmUiRfOfOcK9H6E2B+x(u%rZoWlThQeMbJ8G4D1A-w*t!qYnVjSgPdLaI6F3C0y)v&s#pXlUiRfNcK9H5E2B+x(u$rZoWkThQeMbJ7G4D1z-w*t!qYmVjSgOdLaI6F3B0y)v%s#pXlUiQfQfNcK8H5E2A+x*u$rZnWkThPeMaJ7G4C1z-w&t!pYmVjRgOdL9I6E3B0y(v%s#oXlUiQfNbK8H5D2A+x*u$qZnWkShPeMaJ7F4C1z)w&t!pYmUjRgOcL9I6E3B+y(v%r%r#oXlTiQeNbK8G5D2A-x*t$qZnVkShPdMaI7F4C0z)w&s!pXmUjRf