Plasmid Miniprep

Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis

Definitions cDNA (complementary DNA) cDNA (complementary DNA) DNA copy of a gene that lacks introns and therefore consists solely of the coding sequence. Made by reverse transcription. 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 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 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 BLASTN/BLASTX /ORF Finder/Clustal W Sequence Analysis

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

Theoretical Basis of the Alkaline Lysis Plasmid Miniprep 1. Lyse Cells 2. Separate nucleic acids from other cellular macromolecules 3.Concentrate nucleic acids 4.Separate RNA from DNA

Alkaline/SDS Cell Lysis SDS: anionic detergent causes cell lysis Sodium hydroxide: base, denatures DNA

Selective Precipitation Potassium Acetate IceCentrifugation

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 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= 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

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. a standard curve is made by plotting the molecular weights of the standards and the distance each fragment has migrated from the 2. measuring the distance the unknown fragment migrated from the standards and the distance each fragment has migrated from the 2. measuring the distance the unknown fragment migrated from the well well 3. substituting the distance the unknown migrated into the equation of 3. substituting the distance the unknown migrated into the equation of the line of best fit, and solving for Y (the molecular wt) the line of best fit, and solving for Y (the molecular wt)