HO-17 - BIO 320 lecture 17 Reading: Text chapter 9.1 - 9.3,...

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GGATCC CCTAGG G CCTAG GATCC G 5' 5' 3' 3' 3' 5' 5' 3' 5' 3' 5' 3' BIO 320 lecture 17 Reading: Text chapter 9.1 - 9.3, pp301-324 text problems 9.1-4,6, 9-12, 18, 19 Genet ic te chnolo gy I Re striction enzyme s, recombi nant DNA and cloning; mak ing and screening gene librar ies . Recombinant DNA manipulation depends on enzymes that can cut, join and copy DNA, on vectors into which DNA fragments can be inserted, and on transformation of cells with cloned DNA. GGG CCC CCC GGG Staggered cuts at a restriction enzyme site create complementary "sticky" ends. Sites are usually 4, 6 or 8bp, and symmetric: the top strand, read left to right = the bottom strand, read right to left. 5' 3' ori amp R EcoR1 Sal1 Bam H1 Hind III Xho1 Kpn1 size: about 3kb Size: about 3,000bp multiple cloning site antibiotic resistance gene ( β -lactamase; breaks down ampicillin) origin of replication lacZ gene A plasmid for cloning in E.coli : inserts placed into the multiple cloning site interrupt the lacZ gene. Cells with an intact lacZ gene can metabolize an artificial substrate, X-gal, to produce a blue product. (The lacZ gene on the bacterial chromosome is already mutated.) Other restriction enzymes can generate 3' overhangs. . ..or blunt ends: G ACGTC CTGCA G 5' 3' 3' 5' 5' 3' 5' 3' 5' 3' 5' 3' 5' 3' Restriction enzymes generate specific DNA fragments, by recognizing and cutting specific sequences Plasmid cloning vectors include. .. - an
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HO-17 - BIO 320 lecture 17 Reading: Text chapter 9.1 - 9.3,...

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