1. Isolation of Genomic DNA from Arabidopsis thaliana

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

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

4. Today’s Laboratory Objectives To isolate high quality genomic To isolate high quality genomic DNA from Arabidopsis thaliana DNA from Arabidopsis thaliana To determine the quantity and To determine the quantity and purity of the isolated DNA purity of the isolated DNA

5. Arabidopsis thaliana Small flowering plant- mouse eared cress (Brassicaceae) Small flowering plant- mouse eared cress (Brassicaceae) The primary model organism in 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) Genome sequenced (125 Mb) Many established genetic resources: mutant lines, microarrays, EST databases Many established genetic resources: mutant lines, microarrays, EST databases Widespread distribution in nature Widespread distribution in nature

6. What do we need to do to isolate genomic DNA? Lyse cells Tissue disruption (mortar/pestle) Membrane solubulization (N-lauryl sarcosine) Inactivate nucleases Liquid N 2 EDTA (chelator) Separate DNA from other cellular macromolecules CTAB binds to and precipitates carbohydrates at NaCl concentrations >0.5 M Phenol:chloroform denatures and precipitates proteins Lipids remain in organic (phenol:chloroform) phase RNAseA treatment Concentrate nucleic acids (remove carbohydrates) Alcohol/cation precipitation

7. New Techniques/Theoretical Basis Plant tissue disruption by mortar and pestle: Liquid N 2 : -196º C inhibits enzymatic (nuclease) activity Physical crushing of plant tissue; cells broken The finer the grind, the better the DNA yield

8. New Techniques/Theoretical Basis Phenol:Chloroform extraction: Phenol:Chloroform (1:1)- organic solvents that denature proteins Insoluble in water- will phase partition in aqueous solutions (specific gravity =1.27) At pH 7.8-8.0, lipids partition in organic phase, nucleic acids partition in aqueous phase, precipitated proteins and carbohydrates are at the interface Phenol is toxic and highly corrosive!

9. 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 Cesium Chloride Gradient

10. 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 A 260 /A 280  Pure DNA A 260 /A 280 = 1.6-1.8

11. Next Week Assess the integrity of the isolated DNA by agarose gel electrophoresis Assess the integrity of the isolated DNA by agarose gel electrophoresis Digest the genomic DNA with Digest the genomic DNA with restriction enzymes

12. Restriction Enzymes  becterial 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)

13. Type II Restriction Enzymes Hundreds of restriction enzymes have been identified. Hundreds of restriction enzymes have been identified. Most recognize and cut palindromic sequences Most recognize and cut palindromic sequences Many leave staggered (sticky) ends Many leave staggered (sticky) ends Important for molecular biologists because restriction enzymes create unpaired "sticky ends" which anneal with any complementary sequence Important for molecular biologists because restriction enzymes create unpaired "sticky ends" which anneal with any complementary sequence

14. Using Restriction Enzymes The activity of restriction enzymes is dependent upon precise environmental conditions: pHTemperature 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

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