基因工程第6章大分子的分离与分析

1、chapter 6 isolation and analysis of macromoleculese. coli plasmid dnaisolation, detection and purification of dnaminipreparation principle under alkaline condition, linear dna denature and form precipitate with cell components, but plasmid dna keep circular, re-nature under high concentration of sal

2、t to retain in the supernatant. steps cell growth harvest and lysis plasmid dna purification（phenol/chloroform, gradient centrifugation）sol i tris-hcl, edta, glucose sol ii naoh-sdssol iii 3m kac edta：mg2+，dnase inhibitorsds：resolve membrane protein and lipid to break cell membrane; resolve nuclear

3、membrane and nucleosome to release nucleic acid; partially inhibit rnase and dnase; denature protein.dna electrophoresissize, concentrationagarose gelagarose: a type of polymernormal melting point (65)low melting point ( 25 20-30) l voltage mobility voltage 2kb 5 u/cml electric field negative positi

4、ve puls field for over hundreds of kbsl temperature 0.5% 4-30 low melting point 4 l buffer tae tbe tpe 50mm (ph7.58.0)alkaline agarose electrophoresis for ssdna or southern blot l loading buffer 溴酚蓝/二甲苯菁ff 40% sucrosepolyacrylamide gel electropholesis（page）advantages: high resolution ( 1 bp) large s

5、ample loading (10g) high purity of recovered samplenon-denature: mobility (sequence and composition)denature (尿素/甲醛): isolate probe analyze the products of enzyme s1 dna sequencingpurification of dna fragment (from agarose）llow melting point agarose gel + 5vol tris 65 5min phenol extract ethanollper

6、meation bag (electrical elution) for 5kblglass milk (bead) 3m nai，bead attachment elutelkit qiagen corp.la typical mammal cell: 10-5g rna，but 80-85% is rrna(28s,18s,5.8s and or 5s (23s,16s,5s),and 1520% consist of small rna（e.g. trna，hsrna etc.）.l15% of total rna is mrna: 3 end poly (a) tail adhere

7、to oligo (dt)-celluloseisolation, purification and detection of rnacontrol of rnase activityexperimental tools and solution glass, plastics, electrophoresis apparatus 180 8hr or longer， chloroform washing/naoh 0.1% depc(焦碳酸二乙酯)incubation(37, overnight) (strong inhibitor of rnase, but not absolutely)

8、 depc-treated water washing and autoclavingelectrophoresis apparatus: lrunning water washing, ethanol drying 3%h2o210min，0.1% depc h2o washinglh2o：0.1% depc （some agents cant be treated by depc, e.g. tris）lothers such as gloves, clean roomextraction of rnalreferencelkit gibco/tr1zol reagent 100ml 99

9、.8usd (酚+异硫氰酸胍) qiagen/rnaeasypurification of rnalremoving protein lremoving dna dnase i （rnase free） lkitlgradient centrifugationpurification of mrnalaffinity chromagraphylpurity detection od260: 1=40g/ml rna od280, od230electrophoresis lagrose gel 氢氧化甲基汞 甲醛 乙二醛-二甲基亚矾（dmso）lpagelprotein sizeslweste

10、rn blotln-terminal amino acid sequencingsds-page of proteinmolecular hybridization(分子杂交分子杂交)typessouthern,northern,western,dot,colony(plaque)ldenature(变性变性) hot, extreme ph, orgonic agents, urealannealing(复性复性) complementary ss annealing to form dslhybridization (杂交杂交) homologous ss annealing to for

11、m dssouthern blotdna fragment blotlre digestionlelectrophoresis/photo neutralalkaline（0.4n, naoh）ldenature/neutralizeblotting nitrocellulose membrane nc(80-100g/cm) nylon membrane (charged or not)(350-500 g/cm) 6ssc(氯化钠和柠檬酸钠) transfer buffer 毛细管 电转移 真空(真空转移装置)滤纸凝胶滤膜吸水纸玻璃板重物支持物500g 1kb 15kb 18hrcross

12、 linking 80 2hr, uv, microwavehybridization prehybridization (预杂交)6sscdenhardt(50 x)：(with ficoll,聚乙烯比咯烷酮)denatured fish sperm dna0.5% sdshybridizationl probe treatmentl 42 50% 甲酰胺l 6-8 hrs probe：dna, oligo, random dnamembrane washing(洗去未杂交的探针) 2ssc/0.5% sds 15min rt 0.1ssc/0.5 % sds 1hrdetection x-

13、film or biochemical methods probe labeling evenly labeled (均一标记) nick translation(切口平移): e.coli dna poly i (dnasei:14-16 to generate nick) random primer: (已取代前者) 6nt or 612nt klenow ss probe ssdna template, universal primer (not like cdna, rnase h) rna probe，rna polymeraseend labeling klenow 3 end,

14、补平和置换 t4 polymerase 3end strong 3-5 exonuclease, esp. for 3 protruding end kinase 5end 正反应 32p5-oh nnnn5pnnnn 交换反应 over atp 使5-padp then 32p5oligo nucleotide kinase, terminal transferaselabels radio: 32p，33p，35s non-radio: digoxigenin, biotin to label dutpexample: dig label/detectionlabelling probe6

15、 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bcip：5-溴-4-氯-3吲哚磷酸nbt: 氯化硝基四氮唑蓝（染料色素） cip的底物, 也称氮蓝四唑优点：无污染，方便，探针可重复使用，可控制显色反应，保存杂交膜，易辩别真假阳性缺点：灵敏度不够，不能多次杂交， 0.1pg可检测单拷贝，如g人胎盘dn

16、a中的单拷贝in situ hybridization (原位杂交) (colony or plaque)colony growth positive clones two plates (有/无膜)replicating most colony or plaque is recombinants (white color)dna release and crosslinking many methods 10%sds； 0.5n naoh/1.5n nacl 0.5m tris 1.5n nacl ssc 干燥 固定 80 2hrdot hybridization (斑点杂交斑点杂交)dir

17、ectly loading dna sample on membranenorthern blot similar to southern blot, detecting total rna or mrna.electrophoresis bufferl 甲醛 buf (5) 0.1mm mops(ph2.0) (3-(n-玛琳代)丙磺酸) 40mm naac 5mm edta(ph8.0) depc h2ol gel making 1buf + 2.2m 甲醇 （mw 30.03, 37% 水溶液，12.3m（ph4.0）loadingrna treatment (up to 30g）4.5

18、l+5buf 2.0l 甲醛 3.5l 甲酰胺 10l 65 15min loading buffer 50% 甘油，1mm edta(ph8.0) 0.25% bpb 0.255二甲苯tffrunning geltransfer similar to southern blotwestern blotsimilar to the blot above mentionedkey difference: antibody as probemonoclonal antibody specificpolyclonal mixed antibodiessteps:lprotein preparatio

19、nlrunning sds-pageltransferring (nitrocellulose membrane)lstainingl blocking fat-free milk 5%l first antibody bind target proteinl secondary immunological reaction second antibody(抗免疫球蛋白抗体) protein a labels：125i 碱性磷酸酶 辣根过氧化物酶ldetectionrna terminus analysislenzyme s1 mapping(参见分子克隆指南,第558页)labeled oligo, page gel to determine mw53atguags1酶s1酶detecting splicing si