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Plasmid Miniprep Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis Definitions cDNA (complementary DNA) DNA copy of a gene that lacks introns and therefore consists solely of the coding sequence. Made by reverse transcription. cDNA Library Collection of genes in their cDNA form, lacking introns cDNA Library Construction Laboratory Objective To isolate plasmid containing a cDNA insert from Emiliania huxleyi, to use as a template in a DNA sequencing reaction Research Plan Preparation of Competent Cells/Bacterial Transformation Growth of Transformant and Plasmid MiniPrep DNA Sequencing Sequence Analysis BLASTN/BLASTX /ORF Finder/Clustal W Today’s Laboratory Objectives To isolate high quality plasmid DNA that can be used as template for DNA sequencing To quantify and determine the purity of the isolated plasmid DNA To determine the size of the plasmid DNA and its insert Map of Parent Vector pMAB58 SnaBI BspEI SwaI PpuMI XbaI Bsu36I ARS4/Cen6 TRP1 7000 DraIII Amp AhdI 1000 F1 6000 BamHI pT7 7577 bps Ter ADH attB2 pMAB58 2000 ori 5000 ccdB 3000 SacII ++ AatII 4000 attB1 BsrGI MluI AD NLS NotI pADH EcoRI BsaBI SmaI BsrGI BsmI XbaI EcoRI PacI MluI BsrGI SexAI BseRI Eco47III RsrII NspV BsaBI Asp718I KpnI ApaI Bsp120I StyI BsmI AlwNI Theoretical Basis of the Alkaline Lysis Plasmid Miniprep 1. 2. 3. 4. Lyse Cells Separate nucleic acids from other cellular macromolecules Concentrate nucleic acids Separate RNA from DNA Alkaline/SDS Cell Lysis SDS: anionic detergent causes cell lysis Sodium hydroxide: base, denatures DNA Selective Precipitation Potassium Acetate Ice Centrifugation Separation of Nucleic Acids RNase PEG Precipitation For Cleaner DNA Precipitate w/ Polyethylene Glycol and NaCl Theoretical Basis of UV Spectrophotometry A UV spectophotometer measures the amount of light particular molecules absorb (Proteins at A280; Nucleic Acids at A260) Lambert-Beer law describes the relationship between absorptivity coefficient and concentration and is given by the following equation: A=εbc Where: b= light path length c=concentration of substance ε=extinction coefficient For DNA the extinction coefficient, ε= 1/50 ug/ml Theoretical Basis of UV Spectrophotometry To Quantify your DNA sample: A260 x Dilution Factor x 50 ug/ml= concentration of nucleic acids in a sample using a 1 cm pathlength To estimate the purity of your sample: A260/A280= ratio of nucleic acids/protein A260/A280= 1.6-1.8 is optimal for DNA Theoretical Basis of Agarose Gel Electrophoresis Agarose is a polysaccharide from marine alage that is used in a matrix to separate DNA molecules Because DNA ia a (-) charged molecule when subjected to an electric current it will migrate towards a (+) pole Pouring an Agarose Gel 1 2 4 5 7 8 3 6 9 Sizing a Piece of DNA The distance the DNA migrates is dependent upon the size of the DNA molecule the secondary structure of the DNA the degree of crosslinking in the gel matrix Size of DNA molecule can be determined by using standards of known molecular weight 1. 2. 3. a standard curve is made by plotting the molecular weights of the standards and the distance each fragment has migrated from the measuring the distance the unknown fragment migrated from the well substituting the distance the unknown migrated into the equation of the line of best fit, and solving for Y (the molecular wt)