recombinantdnaoutline

recombinantdnaoutline - RECOMBINANT DNA OUTLINE A....

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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
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3. Procedure Highlights 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
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recombinantdnaoutline - RECOMBINANT DNA OUTLINE A....

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