基因工程 第6章 大分子的分离及分析.ppt

Oligo nucleotide Kinase, Terminal transferase Labels Radio: 32P，33P，35S Non-radio: DIGoxigenin, Biotin to label dUTP Example: DIG label/detection Labelling probe 6 Nt oligo primer (Klenow) Detection washing blocking immunological reaction (labeled Anti-DiG-Ab-Ap (碱性磷酸酶) washing color developing/chemiluminescence (化学发光) NBT-BCIP 形成棕黄色沉淀 * Chapter 6 Isolation and analysis of macromolecules E. Coli plasmid DNA Isolation, detection and purification of DNA Minipreparation ① principle Under alkaline condition, linear DNA denature and form precipitate with cell components, but plasmid DNA keep circular, re-nature under high concentration of salt to retain in the supernatant. ② Steps cell growth harvest and lysis plasmid DNA purification（phenol/chloroform, gradient centrifugation） Sol I Tris-HCl, EDTA, Glucose Sol II NaOH-SDS Sol III 3M KAc EDTA：Mg2+，DNase inhibitor SDS：resolve membrane protein and lipid to break cell membrane; resolve nuclear membrane and nucleosome to release nucleic acid; partially inhibit RNase and DNase; denature protein. DNA electrophoresis Size, concentration Agarose gel Agarose: a type of polymer Normal melting point (?65℃) Low melting point ( 25℃ 20-30℃) voltage mobility voltage ?2kb ? 5 U/cm electric field Negative → positive puls field for over hundreds of kbs temperature 0.5% 4℃-30℃ low melting point 4℃ buffer TAE TBE TPE 50mM (pH7.5～8.0) Alkaline agarose electrophoresis for ssDNA or Southern blot loading buffer 溴酚蓝/二甲苯菁FF 40% Sucrose Polyacrylamide gel electropholesis（PAGE） Advantages: high resolution ( 1 bp) large sample loading (10?g) high purity of recovered sample Non-denature: mobility (sequence and composition) Denature (尿素/甲醛): isolate