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2 DNA DNA DNA DNA

3 4 DNA A DNA B DNA C

4 4 DNA

5 4 DNA A DNA

6 GAATTC CTTAAG GAATTC CTTAAG EcoRI G AATTC G AATTC CTTAA G CTTAA G G AATTC G AATTC CTTAA G CTTAA G G AATTC G AATTC CTTAA G CTTAA G T4-DNA ligase GAATTC CTTAAG GAATTC CTTAAG GAATTC CTTAAG GAATTC CTTAAG

7 TGATCA ACTAGT GGATCC CCTAGG GGATCC CCTAGG BclI BamHI T GATCA ACTAG T GATCC G G CCTAG T GATCC G GATCA ACTAG G CCTAG T T GATCC G GATCA ACTAG G CCTAG T T4-DNA ligase TGATCC ACTAGG TGATCC ACTAGG GGATCA CCTAGT GGATCA CCTAGT

8 CTGCAG GACGTC GGATCC CCTAGG GGATCC CCTAGG PstI BamHI CTGCA 3' G G 3' ACGTC GATCC G G CCTAG T4-DNA pol Klenow C G G C GATCC CTAGG GGATC CCTAG T4-DNA ligase CGATCC GCTAGG CGATCC GCTAGG GGATCG CCTAGC GGATCG CCTAGC

9 5 G AATTC EcoR I CTTAA G GATCC G G CCTAG BamH I Klenow Klenow GAATT AATTC CTTAA TTAAG GATCC CTAGG GGATC CCTAG TdT dgtp TdT dctp GAATTGGGGGG AATTC CTTAA GGGGGGTTAAG GATCC CCCCCCCTAGG GGATCCCCCCC CCTAG T4-DNA ligase BamH I GAATTGGGGGGGATCC CTTAACCCCCCCTAGG BamH I GGATCCCCCCCAATTC CCTAGGGGGGGTTAAG GAATTGGGGGGGATCC CTTAACCCCCCCTAGG GGATCCCCCCCAATTC CCTAGGGGGGGTTAAG

10 3 GCATG 3' C C 3' GTACG Sph I G 3' ACGTC CTGCA 3' G Pst I TdT dctp TdT dgtp GCATGCCCCCC 3' C C 3' CCCCCCGTACG G 3' GGGGGGACGTC CTGCAGGGGGG 3' G GCATGCCCCCC G C GGGGGGACGTC GCATGCCCCCC G C GGGGGGACGTC CTGCAGGGGGG C G CCCCCCGTACG CTGCAGGGGGG C G CCCCCCGTACG Pst I Klenow GCATGCCCCCCTGCAG CGTACGGGGGGACGTC GCATGCCCCCCTGCAG CGTACGGGGGGACGTC T4-DNA ligase Pst I CTGCAGGGGGGCATGC GACGTCCCCCCGTACG CTGCAGGGGGGCATGC GACGTCCCCCCGTACG

11 G 3' C C 3' G 3' G C C 3' G TdT dttp TdT datp GTTTTTTTTTT 3' C C 3' TTTTTTTTTTG G 3' AAAAAAAAAAC CAAAAAAAAAA 3' G T4-DNA ligase GTTTTTTTTTTG CAAAAAAAAAAC GTTTTTTTTTTG CAAAAAAAAAAC CAAAAAAAAAAC GTTTTTTTTTTG CAAAAAAAAAAC GTTTTTTTTTTG GT TTTTTTT TTG CAA AAAAA A AAC CAA AAAAA A AAC GT T TTTTT T TTG S1 TG AC CA GT

12 BamHI GGATCC CCTAGG G GATCC CCTAG G Klenow GGATC GATCC CCTAG CTAGG T4-DNA ligase EcoR I GGAATTCC CCTTAAGG EcoR I linker GGATCGGAATTCCGATCC CCTAGCCTTAAGGCTAGG

13 DNA 25-75% DNA

14 DNA5 : 1-10 : 1 DNA GAATTC CTTAAG GAATTC CTTAAG EcoRI G p AATTC G HO AATTC CTTAA OH G CTTAA p G HO OH G-A-A-T-T-C G A-A-T-T-C C-T-T-A-A G C-T-T-A-A-G HO OH

15 GCATG 3' C C 3' GTACG G 3' ACGTC CTGCA 3' G TdT dctp TdT dgtp GCATGCCCCCC 3' C C 3' CCCCCCGTACG G 3' GGGGGGACGTC CTGCAGGGGGG 3' G GCATGCCCCCC G C GGGGGGACGTC CTGCAGGGGGG C G CCCCCCGTACG Klenow T4-DNA ligase GCATGCCCCCCTGCAG CGTACGGGGGGACGTC CTGCAGGGGGGCATGC GACGTCCCCCCGTACG

16 4 DNA B DNA

17 Ca 2+ Ca Ca 2+

18 Ca ml OD 600 = ml 10 mm CaCl 2 1 ml 75 mm CaCl

19 Ca µl 50 ng DNA ml 37 1

20 DNA DNA

21 34% 10.3%

22 0.2-1ml µl DNAPEG1000Ca 2+

23 λdna MgCl 2 OD 600 = 0.5

24 DNA DNA

25 DNA DNA DNA

26 puc puc puc183.4x X10 17 / 2686X pUC18 puc182ml 2X pUC18 DNA

27 DNA DNA20%10 7 µg 100 DNA 100% / 10 7 X 10-2 = 0.1 µg DNA 20% 0.1 / 20% = 0.5 µg DNA DNA101 5 µg

28 DNA

29 pbr322 JM83ED8767

30 Ca ml 50 ng DNA ng DNA Ca 2+ λ-dna / µg DNA / µg DNA / µg DNA / µg DNA

31 Ca λ30

33 4 DNA C DNA

34 4 DNA C

35 DNAEcoRI Ap r Tc r Ap r Tc r DNABamHI Ap r Tc r Ap r Tc s Pvu I Pst I ori Ap r Pst I ROI EcoR I Cla I Hind III pbr bp ROP Tc r Hind II BamH I Sal I Bal I

36 Ap Ap Ap ApTc ApApTc Ap + Tc

38 trp + tktk - dttp AP AP dcdp dtdp dttp TK T R TK dtmp dtdp dttp

39 puc18lacz pij702melc

40 puc18lacz Ap r lacz - Ap r lacz + Ap + X-gal Ap r lacz' puc18 ori Ap r + lacz -

41 DNA DNA

42 DNA 1.0 kb 0.8 kb B E P B BamHIDNA 2.7 kb kb 2.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz 4.0 kb DNA2.7 kb puc kb ori Ap r

43 DNA 1.0 kb 0.8 kb S B P S DNApUC18 SphISphI SphI kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz 50HindIIIEcoRI puc kb ori SphI1 / 50 Ap r

44 DNA 1.0 kb 0.8 kb B E P B EcoRIDNA 3.0 kb kb 1.0 kb kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz 4.0 kb PstIDNA 3.2 kb kb 0.8 kb kb Ap r puc kb ori

45 DNA 2.0 kb 2.0 kb B E E B 4.0 kb DNAEcoRI 2.0 kb2.7 kb 2.0 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz puc kb 2.7 kb ori 2.0 kb Ap r

46 DNA E kb B B B E BamHI kb BamHI+EcoRI kb 1.2 kb BamHI kb 1.4 kb0.6 kb0.8 kb 2.6 kb0.8 kb1.8 kb 4.0 kb0.6 kb3.4 kb 4.4 kb PstI EcoRI BamHI 2.2 kb

47 DNA DNA DNA

48 DNA 0.4NNaOH SSC 80 SSC-SDS X

49 DNA DNA 12 cdna mrna GACCTAAAGCGGATCGTAGGTC GACCTAA A GC G CTGGATG G C TA

50 DNA T4-PNP 5 HO 3 HO OH 3 OH 5 T4-PNP Mg 2+ pppatpγ- 32 P-ATP 5 p 3 HO OH 3 p 5

51 DNA 3 AAAAAAAAAAACCAGCTTCC GAACTGATTTAGGCT 5 mrna 5 TTTTTTTTTTT Mg 2+ dntp + pppdatpα- 32 P-dATP 3 AAAAAAAAAAACCAGCTTCC GAACTGATTTAGGCT 5 TTTTTTTTTTTGGTCGAAGG CTTGACTAAATCCGA 5 mrna 3 cdna

52 DNA DNA 5 G-C-T-C-A-G-C-T-G-G-A-G-T 3 3 C-G-A-G-T-C-G-A-C-C-T-C-A 5 DNase I 5 G-C-T-C A-G-C-T-G-G-A-G-T 3 3 C-G-A-G-T-C-G-A-C-C-T-C-A 5 DNA pol I Mg 2+ 5 dntp 5 ppp daα- 32 P-dATP 5 G-C-T-C-A-G-C-T-G-G-A-G-T 3 3 C-G-A-G-T-C-G-A-C-C-T-C-A 5

53 DNA ABC Biotin GCTTGAGCAGTAACCTG Avidin

54 DNA ABC Biotin GCTTGAGCAGTAACCTG Avidin

55 DNA dutp-- digoxigenin DIG -

56 DNA DNA DNA SangerDNA DNADNA DNAddNTP 600 bp / 601 bp DNAKit

57 DNA DNA DNA DNA A C G T ssdna ssdna ssdna ssdna ssdna ssdna ssdna ssdna Primer Primer Primer Primer Primer Primer Primer Primer Klenow Klenow Klenow Klenow Klenow Klenow Klenow Klenow dntps dntps dntps dntps dntps dntps dntps dntps ddatp ddatp ddctp ddctp ddgtp ddgtp ddttp ddttp A G T C DNA DNA

58 DNA DNA GGCATTGCGTTACTAGTCCAGTACA P OH 3' A C G T Klenow dntp + ddatp P T T T T T T OH 3'

62 DNA 80 DNA

63 DNA DNA DNA DNA mrna mrna mdna mdna

64 I 125 IgG I 125 IgG

65 37

Microsoft PowerPoint - 2014_autumn-molecular biology-08.ppt [兼容模式]

Microsoft PowerPoint - 2014_autumn-molecular biology-08.ppt [兼容模式] 第五章分子生物学研究法 ( 上 ) DNA RNA 及蛋白质操作技术主要内容模式生物核酸的操作和分析技术蛋白质的分离和分析什么是模式生物? 分子生物学有一个著名的说法 : 基础问题可以在最简单和最容易获得的系统中得以回答能够