Isolation of Genomic DNA from Arabidopsis thaliana

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Isolation of Genomic DNA from Arabidopsis thaliana
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Broad and Long Term Objective
To determine the copy number of Myb transcription
factor genes in the genome of the model plant
Arabidopsis thaliana
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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
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Todays Laboratory Objectives

• To isolate high quality genomic
• DNA from Arabidopsis thaliana
• To determine the quantity and
• purity of the isolated DNA

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Arabidopsis thaliana

• Small flowering plant- mouse eared cress
  (Brassicaceae)
• 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)
• Many established genetic resources mutant lines,
  microarrays, EST databases
• Widespread distribution in nature

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What do we need to do to isolate genomic DNA?

• Lyse cells
• Tissue disruption (mortar/pestle)
• Membrane solubulization (N-lauryl sarcosine)
• Inactivate nucleases
• Liquid N2
• EDTA (chelator)

• Separate DNA from other cellular macromolecules
• CTAB binds to and precipitates carbohydrates at
  NaCl concentrations gt0.5 M
• Phenolchloroform denatures and precipitates
  proteins
• Lipids remain in organic (phenolchloroform)
  phase
• RNAseA treatment
• Concentrate nucleic acids (remove carbohydrates)
• Alcohol/cation precipitation

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New Techniques/Theoretical Basis

• Plant tissue disruption by mortar and pestle
• Liquid N2 -196º C inhibits enzymatic (nuclease)
  
• activity
• Physical crushing of plant tissue cells broken
• The finer the grind, the better the DNA yield

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New Techniques/Theoretical Basis

• PhenolChloroform extraction
• PhenolChloroform (11)- 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!

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New 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 buoyant density

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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

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Next Week

• Assess the integrity of the isolated DNA by
  agarose gel electrophoresis
• Digest the genomic DNA with
• restriction enzymes

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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)

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Type II Restriction Enzymes

• Hundreds of 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

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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

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Next Week

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