Transcript Phosphoenolpyruvate carboxykinase

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
Washes and Chemiluminescent Detection
Data Analysis
Today’s Objectives
1. To Isolate High Quality Genomic DNA from Arabidopsis
2. Determine the Quantity and Purity of the Genomic DNA
Arabidopsis Thaliana
The arabidopsis information resource: http://www.arabidopsis.org/
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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
What do we need to do to isolate
genomic DNA?
Techniques/Theoretical Basis
Fundamental Steps in Isolation:
Disrupt tissue
Break open cells
Extract DNA from other cellular components
Precipitate DNA
Grinding in Liquid Nitrogen
Disrupts tissues and facilitates breaking of cells
Techniques/Theoretical Basis
Components of Reaction Buffer
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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
Removal of Cellular Components
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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
Precipitation of DNA
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Accomplished using various salts and ETOH to pull
water away from DNA
Effective means of concentrating DNA
Techniques/Theoretical Basis
Cesium Chloride Gradient
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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
Techniques/Theoretical Basis
Ultraviolet Spectroscopic Analysis of Nucleic Acids
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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
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Assess the Integrity of the isolated DNA by agarose
gel electrophoresis
Restrict the genomic DNA