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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.

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Presentation Transcript
broad and long term objective
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
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

today s laboratory objectives

Today’s 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

spectrophotometric determination of dna concentration purity
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

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

Theoretical Basis of Agarose Gel Electrophoresis
sizing a piece of dna
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
assessing the integrity of dna
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

slide10

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
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
using restriction enzymes
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
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)

next week
Next Week
  • Agarose gel elctrophoresis of

digested DNA

  • Capillary transfer of DNA from the

gel to a nylon membrane

common lab report problems
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?)