Restriction Digestion of
Arabidopsis thaliana
Genomic DNA
Broad and Long Term Objective
To determine the copy number of Myb
transcription factor genes in the genome of
the model plant Arabidopsis thaliana
Research Plan
Isolate Genomic DNA
Southern Blot
Digest Genomic DNA with Various Restriction Enzymes
Agarose Gel Electrophoresis and Southern Transfer
Make Non-Radioactive Myb Probe
Hyribidize Probe to Southern Blot
Washes and Colorimetric Detection
Data Analysis
Today’s Laboratory
Objectives
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To determine the purity and
yield of isolated genomic DNA
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To determine integrity of
isolated genomic DNA
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To digest genomic DNA with
various restriction enzymes
Spectrophotometric determination
of DNA concentration/purity
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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
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DNA concentration (ng/ul) =
A260 (dilution factor) (50 ng/ul)
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Estimate of DNA purity =
A260/A280 ratio (1.6-1.8 is optimal)
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Total DNA yield (ng) =
DNA concentration (volume of water, ul)
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DNA yield per gram tissue =
Total DNA yield/starting weight of A.
thaliana tissue
Theoretical Basis of Agarose
Gel Electrophoresis
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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
Pouring an Agarose Gel
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Sizing a Piece of DNA
Size of DNA molecule can be determined by using
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standards of known size
1. 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
Assessing the Integrity of DNA
High Quality Genomic DNA
>95% 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
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)
Type II Restriction
Enzymes
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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
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
Digesting Genomic DNA for
Southern Blotting
• Restriction sites are located at random in the genome
Myb gene sequence
Mixture of different sized fragments
Separation of fragments by
size (electrophoresis)
Hybridization with myb probe
undigested
Digestion with
EcoRI
EcoRI digested
EcoRI sites
Next Week
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Agarose gel elctrophoresis of
digested DNA
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Capillary transfer of DNA from the
gel to a nylon membrane
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,
experimental; what did you observe; why?)
Specific questions/experiments for future research (given these results, what
is the next step?)