MYB61

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MYB61

• Single or Multicopy gene in Arabidopsis Thaliana?

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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
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Todays Objectives

• 1. To Isolate High Quality Genomic DNA from
  Arabidopsis
• 2. Determine the Quantity and Purity of the
  Genomic DNA

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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

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What do we need to do to isolate genomic DNA?
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Techniques/Theoretical Basis

• Fundamental Steps in Isolation
• Disrupt tissue
• Break open cells
• Extract DNA from other cellular components
• Precipitate DNA

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Grinding in Liquid Nitrogen
Disrupts tissues and facilitates breaking of cells
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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

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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

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Precipitation of DNA

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

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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

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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

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Next Week

• Assess the Integrity of the isolated DNA by
  agarose
• gel electrophoresis
• Restrict the genomic DNA