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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.

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Single or Multicopy gene in Arabidopsis Thaliana?

Research plan

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

Today’s Objectives

1. To Isolate High Quality Genomic DNA from Arabidopsis

2. Determine the Quantity and Purity of the Genomic DNA

Arabidopsis thaliana

Arabidopsis Thaliana

The arabidopsis information resource:

  • 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

What do we need to do to isolate genomic DNA?

Techniques theoretical basis

Techniques/Theoretical Basis

Fundamental Steps in Isolation:

Disrupt tissue

Break open cells

Extract DNA from other cellular components

Precipitate DNA

Grinding in liquid nitrogen

Grinding in Liquid Nitrogen

Disrupts tissues and facilitates breaking of cells

Techniques theoretical basis1

Techniques/Theoretical Basis

Components of Reaction Buffer

  • 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

Removal of Cellular Components

  • 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

Precipitation of DNA

  • Accomplished using various salts and ETOH to pull water away from DNA

  • Effective means of concentrating DNA

Techniques theoretical basis2

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 boyant density

Techniques theoretical basis3

Techniques/Theoretical Basis

Ultraviolet Spectroscopic Analysis of Nucleic Acids

  • 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

Next Week

  • Assess the Integrity of the isolated DNA by agarose

    gel electrophoresis

  • Restrict the genomic DNA

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