Lecture2 - Purify recombinant DNA E.coli...

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MCB 110L Spring 2011 Lecture 2 1/21/2011 Prof. Qing Zhong 306 Barker Hall
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PCR- one of the most amazing inventions in science Kary Mullis -B.S. Georgia Institute of Technology 1966 -Ph.D. Biochemistry, UC Berkeley, 1972 -Cetus, Emeryville, CA, 1979-1986 -1983 invented PCR -1993 Nobel Prize
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Keys: -Cycling -Thermostable DNA polymerase (Taq) First 3 cycles of PCR
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PCR-Polymerase chain reaction • Thermalcycler -Denaturation: 95 o C 30s -annealing: 65 o C 30s-1min -extension: 72 o C 1min/kb • In a PCR reaction: -Template DNA -dNTP -Primers (at 3’) -DNA polymerase (Taq) (5’ to 3’) -buffer Watch this video about PCR: http://www.youtube.com/watch?v=j9Gu7iwBi4I
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Recombinant DNA The Process for producing biologically functional molecular chimeras which could not exist in nature. Restriction endonucleases is the key
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DNA cloning -Enzyme digestion -ligation -In frame
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A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmid DNA
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Unformatted text preview: Purify recombinant DNA E.coli E.coli-Gram-negative 2.5uM 4.6million bps 4000 genes-easy to grow 20 min/cycle-no nuclear membrane K-12-Bacterial restriction modifcation removed-DNA recombination system modifed-endonuclease activity mutated Lac operon - a beautiful example of gene expression Lac operon- a promoter, a transcriptional regulatory site (operator), three structure genes Inducers of Lac operon - inactivate repressor IPTG-Non-hydrolyzable analog of allolactose Nature inducer Direct inducer-intermediate metabolite of lactose IPTG inducible system Widely used to express recombinant proteins. X-gal is cleaved by -galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product. X-gal reaction-a colorful marker Blue-white selection Blue: no insertion White: insertion Insertion leads to disruption of -galactosidase...
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This note was uploaded on 09/12/2011 for the course MCB 120L taught by Professor Fairclough during the Spring '08 term at UC Davis.

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Lecture2 - Purify recombinant DNA E.coli...

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