分子生物学实验技术总汇课件.ppt

第二章 分子生物学方法Chapter 2 Methods of Molecular Biology;Methods of Molecular Biology;2.1 Molecular Separations（分子的分离）;聚丙烯凝胶电泳分辨率高，适合分辨较小的DNA或蛋白质； 琼脂糖凝胶电泳分辨率较低，适合分离较大的核酸； 极长的DNA（30~50kb）需要用脉冲场凝胶电泳； ;;脉冲场（pulsed-field）凝胶电泳;SDS-polyacrylamide gel electrophoresis ;常用蛋白质分子量标准参照物 ;用双向电泳和质谱分析进行蛋白质组分析; Two-dimensional gel electrophoresis; Ion-exchange chromatography（离子交换层析）;Multiple forms of eukaryotic RNA polymerase 真核生物的多种RNA聚合酶的分离; Gel filtration chromatography;Affinity chromatography(亲和层析);2.2 Molecular Cloning Methods（分子克隆方法）;See Molecular Biology page 61; Cutting 切割 Linkage 连接 Replication 复制 Transfer 转移 Restriction endonuclease （RE）限制性内切酶 Nuclease 核酸酶 Ligase 连接酶 Polymerase 聚合酶 Phosphatase and kinase 磷酸化酶和激酶 Methylase 甲基化酶;;See 656;;概念;HindII recognizes this sequence: ? GTPyPuAC Pu: purine (A or G) CAPuPyTG Py: pyrimidines (T or C) ? 4 bp sequence 44 = 256 bases 6 bp sequence: more common 46 = 4096 bases 8 bp sequence 48 = 65000 bases (NotI);Isocaudamer 同尾酶，产生相同的粘性末端 BamHI G?GATCC BalII A?GATCT Isoschizomer 同裂酶，识别相同的DNA序列 XmaI C?CCGGG SmaI CCC?GGG ;Examples Able was I ere I saw Elba (Napoleons lament) Straw? No, too stupid a fad; I put soot on warts, ( a wart remedy) Go hang a salami! Im a lasagna hog. (a statement of preference in Italian food) ;EcoRI ;blunt end 平头末端 sticky end 粘性末端 ;计算题;A piece of DNA (a plasmid or a phage DNA) that serves as a carrier in gene cloning experiments. Vectors fall into two major classes: ;Plasmids as Vectors;a) 分子量小，3-10kb,是一个松弛型控制的复制子；; b) Containing only one cutting site for each of restriction enzymes.只有一个限制性核酸内切梅； c) Easy to be detected（易发现）.;克隆外部DNA使用含有PstI 位点的f pBR