RecombinantDNA technology

RecombinantDNA technology - Recombinant DNA Technology...

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Recombinant DNA Technology Chapter 18 Basic Techniques Used to Clone Genes 0. Isolate DNA of interest 1. Insertion into self replicating vector 2. Expression of gene Discoveries 0. Vectors 1. Enzymes 0. restriction enzymes 1. ligase 2. Gel Electrophoresis 3. Blots 4. Sequencing 5. Fingerprinting Restriction Enzymes 6. First isolated from bacteria 7. Homodimers 8. Recognize palindromic sequences 9. Three different classes 2. Type I 3. Type II 4. Type III 10.Over 3000 have been isolated 5. 800+ commercially available Recognition Sites 3. 4 bp 4. 6 bp 5. 8 bp 6. Frequency of sites in random sequence: 1/4 4 = 1/256 so every 256 bases, would expect to see a restriction site for an enzyme that has a 4 base recognition sequence 1/4 6 = 4096 1/4 8 = 65,536 Generated Ends 7. 5’ (always a phosphate group) 8. 3’ (always OH group)
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9. If 5’ or 3’ overhang - “sticky ends” 10.Blunt ends Agarose Gel Electrophoresis 11.“carrying with electricity” 12.Polysaccharide 13.Application of current 0. DNA is negatively charged 1. Moves toward positive electrode 2. Rate of migration is inversely proportional to the log 10 of MW 3. Other factors 0. Shape of DNA 1. Concentration of agarose 2. presence of EtBr Visualizing DNA 11.Ethidium bromide (EtBr) 12.Intercalates btw DNA base pairs 13.Excite with UV light and it emits light in the visible range 14.**mutagenic** 15.Can detect as little as 1 - 5 - 10 ng of DNA 16. Use ~ 1 μ g marker containing known fragment sizes 17.Stain intensity is proportional to amt of DNA present. Cloning Vectors 14.Three essential components 4. an origin of replication 5. selectable marker gene 4. usually drug resistance to host cell 6. unique restriction cleavage site
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This note was uploaded on 05/01/2008 for the course BSCI 222 taught by Professor Gdovin during the Spring '08 term at Maryland.

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RecombinantDNA technology - Recombinant DNA Technology...

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