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Isolation of Genomic DNA from Arabidopsis thaliana Broad and Long Term Objective Research Plan Today’s Laboratory Objectives To isolate high quality genomic DNA from Arabidopsis thaliana To determine the quantity and purity of the isolated DNA Arabidopsis thaliana Small flowering plant- mouse eared cress (Brassicaceae) The primary model organism in plant biology (small stature, 45 day generation time, high seed yield, simple genome, easy to transform) Genome sequenced (125 Mb) Many established genetic resources: mutant lines, microarrays, EST databases Widespread distribution in nature What do we need to do to isolate genomic DNA? New Techniques/Theoretical Basis New Techniques/Theoretical Basis New Techniques/Theoretical Basis Used to obtain highly pure DNA DNA in gradient subjected to centrifugal force of 105,000 xg DNA forms band in gradient at its buoyant density Spectrophotometric determination of DNA concentration/purity Nucleic acids absorb light at 260 nm Proteins absorb light at 280 nm Purity of Nucleic Acid indicated by A260/A280 Pure DNA A260/A280 = 1.6-1.8 Next Week Assess the integrity of the isolated DNA by agarose gel electrophoresis Digest the genomic DNA with restriction enzymes Type II Restriction Enzymes Hundreds of restriction enzymes have been identified. Most recognize and cut palindromic sequences Many leave staggered (sticky) ends Important for molecular biologists because restriction enzymes create unpaired sticky ends which anneal with any complementary sequence Using Restriction Enzymes The activity of restriction enzymes is dependent upon precise environmental conditions: pH Temperature Salt Concentration Ions One enzymatic unit (U) is defined as the amount of enzyme required to completely digest 1 ug of DNA in 1 hr at 37o C: 3-5 U/ug of genomic DNA 1 U/ug of plasmid DNA Stocks typically at 10 U/ul Next Week Agarose gel elctrophoresis of digested DNA Capillary transfer of DNA f