- RECOMBINANT DNA OUTLINE A Transformation of E.coli cells 1 E.coli well understood cheap numerous a single cell organism b circular

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RECOMBINANT DNA OUTLINE A. Transformation of E.coli cells 1. E.coli – well understood, cheap, numerous a.) single cell organism b.) circular double-stranded DNA chromosome d.) provides enzymes (polymerases, ligases, endonucleases, restriction enzymes) e.) essential in cloning DNA 2. Plasmid a.) extra chromosomal, circular DNA b.) replicates independently of host chromosome (genome) d.) incorporation of foreign DNA 1. restriction enzymes 2. sticky ends 3. ligase 3. Transformation a.) alteration in cell’s genetic makeup caused by introducing exogenous DNA b.) divalent cation (CaCl 2 ) method with Heat Shock 1. binds phosphate groups on lipids 2. neutralizes negative charges 3. heat shock – phase change or thermal distortion – DNA “sneaks” in c.) exponential growth d.) strain preference 4. Procedure Highlight a.) CaCl 2 b.) 42 O C water bath c.) LB media d.) LB agar with ampicilllin B . Plasmid Miniprep 1. Types of Vectors a.) phages b.) plasmid c.) cosmid 2. Plasmid as Cloning Vector a.) small size b.) defined restriction map c.) limited number of restriction sites d.) selectable genetic markers (antibiotic resistance) e.) independent replication site – ori f.) non-mobilizable – can’t transfer to other bacteria through conjugation 3. Procedure Highlights
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a.) TE buffer with glucose c.) potassium acetate/acetic acid C. 1. Restriction Enzymes (RE) a.) found in bacteria b.) cuts both strands at specific recognition sites c.) staggered or blunt cut d.) 4, 6, or 8 base pair sequence for recognition site e.) doesn’t cut own DNA – methylated via methylase f.) endonucleases g.) named by genus, species, strain, and isolation number/order f.) EcoRI – cuts at GAATTC HindIII – cuts at AAGCTT CTTAAG TTCGAA 2. Restriction Fragments a.) cut pieces of DNA c.) can have “sticky ends” which can reanneal to produce recombinant DNA plasmids 3. Agarose Gel a.) separates DNA fragments b.) concentration of agarose determines separation by size of fragments c.) porous, 3 dimensional matrix in buffer 4. Electrophoresis a.) voltage – optimal – 5V/cm b.) DNA negatively charged so moves toward anode c.) distance traveled is inversely proportional to log MW or base pair number d.) loading buffer – more dense with bromophenol blue indicator 5. Staining Fragments a.) ethidium bromide
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This note was uploaded on 05/03/2008 for the course BIO 2322 taught by Professor Spotswood during the Spring '08 term at The University of Texas at San Antonio- San Antonio.

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- RECOMBINANT DNA OUTLINE A Transformation of E.coli cells 1 E.coli well understood cheap numerous a single cell organism b circular

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