plasmid cloning vectors - 6.1 Cloning vectors based on E....

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Unformatted text preview: 6.1 Cloning vectors based on E. coli plasmids 6.1.1 The nomenclature of plasmid cloning vectors pBR322 n 9ª ¸ ™ š 1.“p” 2.“BR” p ¹ h ± 3.“322” ¨è 9ª ¹ P K * ª ¹ L : 6.1.2 The useful properties of pBR322 1.pBR322 H 9ª ¸ ™™ . 2. àéo xö xö . 3. xö (15 . 1000 . 3000 . ). 6.1.3 The pedigree of pBR322 6.1.4 Other typical E. coli plasmid cloning vectors pBR327 . pBR327 . pBR322 k 9ª ¸ ™ œ 9 ª nè¹G P K ¨ 9 ª ¹L 1. . pBR322 . (30 . 45) . 2. Ø ( m fÀ pUC8 . pUC8 . pBR322 lacZ′ . 1. . 500 . 700 . 2. ÌØ 3. DÐ s Î9H¹ª ©E 9ª ¸ ™ œ k 1089bp k ª¸™œ ). Ø ampR . M13mp Ø 6.1.4 Other typical E. coli plasmid cloning vectors 6.1.4 Other typical E. coli plasmid cloning vectors pGEM3Z—— . 9ª DNA ˜ ¸™š 9ª pUC ˜ . ¸™š . ampR . lacZ′ . . lacZ′š ˜ª ¸™ àI ¸ ™ 9˜ª š —— T7 . SP6 . 6.2 Cloning vectors based on M13 bacteriophage @« E 1 ª 507bp H ª¸™• ª ¸ ™ • H4 ª¸™•H ` 5 DNA 5 6.2.1 Development of the cloning vector M13mp2 M13mp7 7 ª ´æ• . EcoRⅠ BamHⅠ SalⅠ 9 ª ´ æ (• M13mp7 . 9ª ´ æ • 7 . DNA . 9ª ´ æ • 7 M13mp7 ˆ ª ´æ• M13mp7 . 9ª ´ æ • ˆ ª ´æ• ˆ DNA . 9ª ´ æ • ˆ 9ª ´ æ 1 ˆ • DNA . 2 . 9ª ´ æ • ˆ 9ª ´ æ 3 ˆ • M13mp7 ˆ 9ª ´ æ • X 9 ª DNA XЩD ¹rˆE EcoRⅠ Recovery of cloned DNA from a recombinant M13mp7 molecule by restriction at the outer sites of the polylinker. M13 . M13mp8 5 M13mp9 8 ª ¸™Ÿ ª ¸™Ÿ 8 , 5 DNA ª ¸ ™ Ÿ 8* 6.2.2 Hybrid plasmid-M13 vectors M13 5 DNA ° 3kb è¸ ™šª o“¨ M13 ° 5 ª DNA è 4¸ ™ š ¹o ± ª¸ ™ š è 5 phagemid . . pEMBL8 è 4ª ¸ ™ š 5 1500bp 6.3 Cloning vectors based on λbacteriophage λ. . 1. . 3kb . 2. ˆ¸ª ™• ™ • ˆ¸ª9 . 6.3.1 Segments of theλgenome can be deleted without impairing viability 15kb 6.3.2 Natural selection can be used to isolate modifiedλthat lack certain restriction sites 6.3.3 Insertion and replacement vectors Щ1E ¨±o¹ª4 . (placement) 5 λ. λ ˜š™¸ª4 (insertion) 5 λ . 5 ˜š™¸ª4 DNA . ¹ P ¨KªhL¹9 n 8kb . EcoRⅠ DNA . ¸ c™ Ⅰ ˜šª9 ˜š™¸ª9 DNA . lacZo ′± ¨ª9 ¹ ЩD E ¸ ™ š ˜ª9 10kb . 6 ±o DNA . 5 t f ¹ªÐ©1E4 . ™ ˜œ¸ª4 λ0 «1E$ @i 7y DNA ˜¸ª4 ™œ DNA ¨¹ª4 P K hLN P K ¨¹ª4 hLN DNA °¹ªÐ©E9 sÅD λEMBL4œ ˜ 9ª ¸ ™ . DNA È . EcoRⅠ BamHⅠ 9 ª ¹ ÀÄsЩED Spi . λ. 20k b SalⅠ λGEM11 . λGEM12 . . 23kb . ª DNA ˜ ¸™œ 9ª ¸ ™ œ ˜ 9ª ¸ ™ 7 ˜ œ ª¸ ™ œ˜ 9ª Sfi™ Ⅰ ˜œ ¸ 9ª ¸ ™ ˜œ Spi . 6.3.4 Cloning experiments withλ insertion or replacement vectors 1. 7 ”æ´ª9 DNA ª ´ (æ ” € X 2. . λ. DNA DNA” 7 æ´ ª . *æ ”ª 7´ *´ (æ ”ª (7 ´ ”ª æ ) 6.3.5 Very large DNA fragments can be cloned using a cosmid (cosmid) . . 9 K ª ¨¹h9 P Lªn 5 5 P304 λ5 cos o 40kb 5 DNA 6.3.5 Very large DNA fragments can be cloned using a cosmid 1. h DNA 2. h„ . 3. . 4‚¶. ¨9 ª 4 5. . ¶ª9 ¨‚4 . ? 6.4 λand other high capacity vectors enable genomic libraries to be constructed M13 .™ Ÿ ȸª9 ™ Ÿ ȸª4 5 5 8kb ȸª9 ™Ÿ 20kb . . 3kb . DNA ȸª4 ™Ÿ λEMBL4 5 . 40kb . 5 5 genomic library . 9DÐ ¹ o M Ð E ©ª P306 oMÈ 6.4 λand other high capacity vectors enable genomic libraries to be constructed N= ln(1 − P) a ln(1 − ) b 9¸ ™a ¸ ªŸ DNA . 9¸ ™N ¸ ªŸ . 9¸ ™ b ¸ ª Ÿ 9¸ ™P ¸ ªŸ ™ª÷ Ÿ¸ /bp 17kb 4.6×106 1.8×107 1.2×108 5.7×108 3.2×109 2.3×1010 820 3225 21500 100000 564000 4053000 35kb 410 1500 10000 49000 274000 1969000 6.5 Vectors for other bacteria ...
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