1 2 3 4 5 6 7 8 9 DNA DNA DNA DNA
4 DNA A DNA B DNA C
4 DNA
4 DNA A DNA
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
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
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
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
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
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
BamHI GGATCC CCTAGG G GATCC CCTAG G Klenow GGATC GATCC CCTAG CTAGG T4-DNA ligase EcoR I GGAATTCC CCTTAAGG EcoR I linker GGATCGGAATTCCGATCC CCTAGCCTTAAGGCTAGG
DNA 25-75% DNA
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
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
4 DNA B DNA
Ca 2+ Ca 2+ 1970Ca 2+
Ca 2+ 100 ml OD 600 = 0.5 10 ml 10 mm CaCl 2 1 ml 75 mm CaCl 2 12-24
Ca 2+ 100 µl 50 ng DNA 2 1-2 1 ml 37 1
DNA DNA
34% 10.3%
0.2-1ml 10-20 µl DNAPEG1000Ca 2+
λdna MgCl 2 OD 600 = 0.5
DNA DNA
DNA DNA DNA
puc1810 8 puc18 10 8 puc183.4x10 11 6.02X10 17 / 2686X6603400pUC18 puc182ml 2X10 10 200pUC18 DNA
DNA DNA20%10 7 µg 100 DNA 100%10 4 10 4 / 10 7 X 10-2 = 0.1 µg DNA 20% 0.1 / 20% = 0.5 µg DNA DNA101 5 µg
DNA
pbr322 JM83ED8767
Ca 2+ 0.1 ml 50 ng DNA 10 9 50 ng DNA Ca 2+ λ-dna 10 6-10 7 / µg DNA 10 5-10 6 / µg DNA 10 7-10 8 / µg DNA 10 6-10 9 / µg DNA
Ca 2+ 37 λ30
4 DNA C DNA
4 DNA C
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 pbr322 4363 bp ROP Tc r Hind II BamH I Sal I Bal I
Ap Ap Ap ApTc ApApTc Ap + Tc
trp + tktk - dttp AP AP dcdp dtdp dttp TK T R TK dtmp dtdp dttp
puc18lacz pij702melc
puc18lacz Ap r lacz - Ap r lacz + Ap + X-gal Ap r lacz' puc18 ori Ap r + lacz -
DNA DNA
DNA 1.0 kb 0.8 kb B E P B BamHIDNA 2.7 kb + 4.0 kb 2.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz 4.0 kb DNA2.7 kb puc18 2.7 kb ori Ap r
DNA 1.0 kb 0.8 kb S B P S DNApUC18 SphISphI SphI100 4.0 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz 50HindIIIEcoRI puc18 2.7 kb ori SphI1 / 50 Ap r
DNA 1.0 kb 0.8 kb B E P B EcoRIDNA 3.0 kb + 3.7 kb 1.0 kb + 5.7 kb HindIII SphI PstI SalI BamHI SmaI KpnI EcoRI lacz 4.0 kb PstIDNA 3.2 kb + 3.5 kb 0.8 kb + 5.9 kb Ap r puc18 2.7 kb ori
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 puc18 2.7 kb 2.7 kb ori 2.0 kb Ap r
DNA E 1.2 0.6 0.8 1.8 kb B B B E BamHI0.6 0.8 1.8 3.4 kb BamHI+EcoRI0.6 0.8 1.8 1.2 2.2 kb 1.2 kb BamHI1.4 2.6 4.0 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
DNA DNA DNA
DNA 0.4NNaOH SSC 80 SSC-SDS X
DNA DNA 12 cdna mrna GACCTAAAGCGGATCGTAGGTC GACCTAA A GC G CTGGATG G C TA
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
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
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
DNA ABC Biotin GCTTGAGCAGTAACCTG Avidin
DNA ABC Biotin GCTTGAGCAGTAACCTG Avidin
DNA dutp-- digoxigenin DIG -
DNA DNA DNA SangerDNA DNADNA DNAddNTP 600 bp / 601 bp DNAKit
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
DNA DNA GGCATTGCGTTACTAGTCCAGTACA P OH 3' A C G T Klenow dntp + ddatp P T T T T T T OH 3'
DNA 80 DNA
DNA DNA DNA DNA mrna mrna mdna mdna
I 125 IgG I 125 IgG
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