分子生物学实验一、实验设计二、实验操作指导教师：林 凤 实验设计基因工程菌的构建 载载 体体 目的基因目的基因酶切PCR扩增，酶切载体片段 目的基因片段+连接重组质粒转化重组子阳性鉴定1、载体多克隆位点2、缓冲溶液3、温度4、末端的性质酶切位点设计载体与目的基因的连接1、粘末端2、平末端3、对照实验连接产物的转化1、核酸电泳检测2、对照实验：空白和阳性阳性重组子的鉴定1、限制性酶切法2、-互补法3、插入失活法4、杂交筛选法 construction of expression vectorNcoI CCATGG GGTACCXhoI CTCGAG GAGCTC实验操作碱裂解法小量制备质粒DNA-pUC18 接种子液至接种子液至75 g/ml Amp75 g/ml Amp的的5 ml LB5 ml LB培养基培养基37 ，OD600 1.0 加氯霉素 100 g/ml 37，过夜1.4 ml培养液4, 6000 rpm, 2 min沉淀加100 l 冰预冷溶液,剧烈振荡弃去上清，尽可能除净培养液室温，20 min200 l 新配制溶液，快速颠倒混匀冰浴5 min150 l 冰预冷溶液，温和振荡冰浴5 min上清加入450 l饱和酚，混匀 4, 12,000 rpm，2 min上清加入450 l氯仿-异戊醇，混匀 4, 12,000 rpm，2 min上清加入300 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，空气干燥10 min20 l TE溶解质粒DNA4, 12,000 rpm，5 minDNA的含量、纯度与分子量的电泳法测定DNA的酶切与连接 酶切反应体系酶切反应体系 1 2 1 2 ddHddH2 2O 19l 19l O 19l 19l 10buffer 2.5l 2.5l 10buffer 2.5l 2.5l 质粒质粒DNA pBR322 3l pUC18 3lDNA pBR322 3l pUC18 3l EcoRI 0.5l 0.5l EcoRI 0.5l 0.5l 37 37 ，1 h1 h 65 65 ，101015 min15 min终止反应终止反应 取取4l 4l 样品，加入样品，加入10Loading buffer 0.5l 10Loading buffer 0.5l ，核酸电泳，核酸电泳l连接反应体系 lddH2O 8.5l l酶切DNA pBR322 20l l酶切DNA pUC18 10llT4 DNA ligase 1l l16 ，15 hl取10l连接产物，核酸电泳（上步剩余酶切产物做对照）l 其余连接产物用于转化重组DNA的转化（一）（一）CaClCaCl2 2法制备大肠杆菌感受态细胞法制备大肠杆菌感受态细胞 0.3 ml 0.3 ml菌种接种于菌种接种于30 ml LB30 ml LB培养基培养基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, 加入1 ml 0.1M CaCl2，分装200 l/管 （二）转化反应分别加入连接产物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 , 90 s冰浴1-2 min弃500 l 上清, 连接产物取100 l , 10 l各涂一平板, 空白，阳性对照和阴性对照200 l各涂一平板加入800 l LB培养基37 水浴5 min37 ，缓慢摇，复苏45 min37 ，12-16 h观察转化子的出现6000 rpm, 5 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