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MYB61

MYB61. Single or Multicopy gene in Arabidopsis Thaliana ?. Research Plan. Isolate Genomic DNA. Southern Blot Analysis. Digest Genomic DNA w/ Various Restriction Enzymes. Agarose Gel Electrophoresis and Southern Transfer. Make Non-Radioactive Myb61 Probe. Hyribidize Probe to Southern Blot.

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MYB61

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  1. MYB61 Single or Multicopy gene in Arabidopsis Thaliana?

  2. Research Plan Isolate Genomic DNA Southern Blot Analysis Digest Genomic DNA w/ Various Restriction Enzymes Agarose Gel Electrophoresis and Southern Transfer Make Non-Radioactive Myb61 Probe Hyribidize Probe to Southern Blot Washes and Chemiluminescent Detection Data Analysis

  3. Today’s Objectives 1. To Isolate High Quality Genomic DNA from Arabidopsis 2. Determine the Quantity and Purity of the Genomic DNA

  4. Arabidopsis Thaliana The arabidopsis information resource: http://www.arabidopsis.org/ • Small flowering plant used as model organism in plant biology • member of the mustard (Brassicaceae) family • Small genome (114.5 Mb/125 Mb total) sequenced in the year 2000 • Extensive genetic and physical maps of all 5 chromosomes • rapid life cycle (6 weeks from germination to mature seed) • Prolific seed production and easy cultivation in restricted space • Efficient transformation utilizing Agrobacterium tumefaciens • A large number of mutant lines and genomic resources • Multinational research community of academic, government and industry laboratories

  5. What do we need to do to isolate genomic DNA?

  6. Techniques/Theoretical Basis Fundamental Steps in Isolation: Disrupt tissue Break open cells Extract DNA from other cellular components Precipitate DNA

  7. Grinding in Liquid Nitrogen Disrupts tissues and facilitates breaking of cells

  8. Techniques/Theoretical Basis Components of Reaction Buffer • Sorbitol= factilitates lysis by increasing osmolarity • EDTA= protect DNA from nucleases by chelating Mg2+ which is required for nuclease activity • Sarcosyl= degergent that disrupts membranes • NaCl/CTAB-cetyltrimethylammonium bromide together w/ sodium chloride facilitate removal of polysaccharides

  9. Removal of Cellular Components • CTAB is a cationic detergent that binds polysaccharides when in solution with NaCl above 0.5 M; CTAB-Polysaccharide complex precipitates during phenol chloroform extraction • Phenol chloroform extraction efficiently denatures proteins and probably dissolves them • RNA removed via enzymatic digestion w/ RNAse

  10. Precipitation of DNA • Accomplished using various salts and ETOH to pull water away from DNA • Effective means of concentrating DNA

  11. Techniques/Theoretical Basis Cesium Chloride Gradient • Used to obtain highly pure DNA • DNA in gradient subjected to centrifugal force of 105,000 xg • DNA forms band in gradient at its boyant density

  12. Techniques/Theoretical Basis Ultraviolet Spectroscopic Analysis of Nucleic Acids • 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

  13. Next Week • Assess the Integrity of the isolated DNA by agarose gel electrophoresis • Restrict the genomic DNA

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