Restriction Digestion of Arabidopsis thaliana Genomic DNA

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Restriction Digestion of Arabidopsis thaliana
Genomic DNA
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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 determine the purity and
• yield of isolated genomic DNA
• To determine integrity of
• isolated genomic DNA
• To digest genomic DNA with
• various restriction enzymes

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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

DNA concentration (ng/ul) A260 (dilution
factor) (50 ng/ul)
Total DNA yield (ng) DNA concentration (volume
of water, ul)
Estimate of DNA purity A260/A280 ratio
(1.6-1.8 is optimal)
DNA yield per gram tissue Total DNA
yield/starting weight of A. thaliana tissue
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Theoretical Basis of Agarose Gel Electrophoresis

• Agarose is a polysaccharide from marine alage
  that is used in a matrix to separate DNA
  molecules
• Because DNA ia a (-) charged molecule when
  subjected to an electric current it will migrate
  towards a () pole

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Pouring an Agarose Gel
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Sizing a Piece of DNA

• Size of DNA molecule can be determined by using
  standards of known size
• A standard curve is made by plotting the size
  (in bp) of the standards (Y-axis) against the
  distance each fragment has migrated from the
  well (X-axis) using semi-log paper
• 2. Measure the distance the unknown fragment
  migrated from the well
• 3. Determine size of the unknown fragment based
  upon the line of best fit by drawing a vertical
  line upward from distance migrated and a
  horizontal line across to the y axis. Report the
  y value (size).
• http//www.csun.edu/vceed002/ref/measurement/data
  /graphpaper/semi_log_numbered.pdf

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Assessing the Integrity of DNA
High Quality Genomic DNA gt95 DNA will be of high
molecular weight, migrating as intact band near
the top of the gel Very little evidence of
smaller fragments indicated by a smear of many
different sized DNA fragments
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Restriction Enzymes

• bacterial 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

• Type II restriction systems endonuclease and
  methylase are separate proteins (binary system)
• Type I, III restriction systems
• endonuclease and methylase in same protein
• Hundreds of type II 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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Digesting Genomic DNA for Southern Blotting

• Restriction sites are located at random in the
  genome

EcoRI sites
Digestion with EcoRI
Myb gene sequence
EcoRI digested
undigested
Mixture of different sized fragments
Hybridization with myb probe
Separation of fragments by size (electrophoresis)
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Next Week

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

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Common lab report problems

• Emiliana huxleyi, Escherichia coli, E. coli not
  E. hux
• Introduction
• Describe experimental details in Materials and
  Methods
• Active voice The E. huxleyi cDNA was
  sequenced, and the sequence
• was analyzed using ORF finder, BLASTN,
• - Materials and Methods
• Volumes not necessary
• Composition and concentration of solutions (what
  is glucose buffer?, what
• concentration of CaCl2?
• Justify methods In order to precipitate the
  nucleic acids in solution, 0.6
• volumes of isopropanol was added to the
  supernatant
• List bioinformatics programs and their uses
  Multiple sequence alignments
• were performed using ClustalW
• Results
• DNA concentration and total yield
• Calculation of transformation efficiencies
• -Discussion
• Do not simply repeat results- analyze your data
  (what were the expected
• results for transformation efficiency of
  control, - control,