Restriction Digestion of Arabidopsis thaliana Genomic DNA

Broad and Long Term Objective To determine the copy number of Myb transcription factor genes in the genome of the model plant Arabidopsis thaliana

Research Plan Isolate Genomic DNA Digest Genomic DNA with Various Restriction Enzymes Agarose Gel Electrophoresis and Southern Transfer Southern Blot Make Non-Radioactive Myb Probe Hyribidize Probe to Southern Blot Washes and Colorimetric Detection Data Analysis

Today’s Laboratory Objectives To determine the purity and yield of isolated genomic DNA To determine integrity of isolated genomic DNA To digest genomic DNA with various restriction enzymes

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 DNA concentration (ng/ul)* = A260 (dilution factor) (50 ng/ul) Total DNA yield (ng)* = DNA concentration (volume of water, ul) Estimate of DNA purity* = A260/A280 ratio (1.6-1.8 is optimal) DNA yield per gram tissue* = Total DNA yield/starting weight of A. thaliana tissue

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 3 1 2 4 5 6 7 8 9

Sizing a Piece of DNA Size of DNA molecule can be determined by using standards of known size* A standard curve is made by plotting the size (in bp) of the standards (Y-axis) against the distance each fragment has migrated from the well (X-axis) using semi-log paper 2. Measure the distance the unknown fragment migrated from the well 3. Determine size of the unknown fragment based upon the line of best fit by drawing a vertical line upward from distance migrated and a horizontal line across to the y axis. Report the y value (size). http://www.csun.edu/~vceed002/ref/measurement/data/graphpaper/semi_log_numbered.pdf

Assessing the Integrity of DNA High Quality Genomic DNA >95% DNA will be of high molecular weight, migrating as intact band near the top of the gel Very little evidence of smaller fragments indicated by a smear of many different sized DNA fragments

Restriction Enzymes bacterial proteins that restrict host range for certain bacteriophages by cleaving specific DNA sequences bacterial “immune system": destroy any "non-self" DNA Self DNA protected by host proteins that methylate the specific DNA sequences recognized by the restriction enzyme (restriction/modification systems)

Type II Restriction Enzymes Type II restriction systems: endonuclease and methylase are separate proteins (binary system) Type I, III restriction systems: endonuclease and methylase in same protein Hundreds of type II 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

Digesting Genomic DNA for Southern Blotting Restriction sites are located at random in the genome EcoRI sites Digestion with EcoRI Myb gene sequence EcoRI digested undigested Mixture of different sized fragments Hybridization with myb probe Separation of fragments by size (electrophoresis)

Next Week Agarose gel elctrophoresis of digested DNA Capillary transfer of DNA from the gel to a nylon membrane

Common lab report problems Emiliana huxleyi, Escherichia coli, E. coli not E. hux Introduction: Describe experimental details in Materials and Methods Active voice: “The E. huxleyi cDNA was sequenced, and the sequence was analyzed using ORF finder, BLASTN,…” - Materials and Methods: Volumes not necessary Composition and concentration of solutions (what is glucose buffer?, what concentration of CaCl2? Justify methods: “In order to precipitate the nucleic acids in solution, 0.6 volumes of isopropanol was added to the supernatant…” List bioinformatics programs and their uses: “Multiple sequence alignments were performed using ClustalW” Results DNA concentration and total yield Calculation of transformation efficiencies -Discussion: Do not simply repeat results- analyze your data (what were the expected results for transformation efficiency of + control, - control, experimental; what did you observe; why?) Specific questions/experiments for future research (given these results, what is the next step?)