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recombinantdnaoutline with answers - RECOMBINANT DNA...

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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 c.) host to plasmids & bacteriophages 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) c.) origin, antibiotic resistance gene & MCS (PCS) 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 b.) alkaline lysis solution – NaOH & SDS c.) potassium acetate/acetic acid d.) isopropanol & ethanol C. Restriction Digestion & Agarose Gel Electrophoresis 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 i.) activity affected by time, temperature, pH & salt concentration 2. Restriction Fragments a.) cut pieces of DNA b.) vary in size according to RE used & locations of cut sites 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 1. planar, organic molecule 2. intercalates the bases in the double helix
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