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Metacaspase. Single or Multicopy gene in Emiliania huxleyi ?. Isolation of Genomic DNA from Emiliania huxleyi. CTAB Isolation and CsCl Gradient. Research Plan. Isolate Genomic DNA. Southern Blot Analysis. Digest Genomic DNA w/ Various Restriction Enzymes.

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Single or Multicopy gene in Emiliania huxleyi?

Isolation of genomic dna from emiliania huxleyi

Isolation of Genomic DNA from Emiliania huxleyi

CTAB Isolation and CsCl Gradient

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 Metacaspase Probe

Hyribidize Probe to Southern Blot

Washes and Chemiluminescent Detection

Data Analysis

Today s objective

Today’s Objective

To Isolate High Quality Genomic DNA from Emiliania huxleyi

Emiliania huxleyi

Emiliania Huxleyi

  • Unicellular eukaryotic marine algae

  • Calcium carbonate coccosphere

  • Blooms of > 100,000 sq kilometers

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

Breaking open E. huxleyi cells

  • SDS

  • Proteinase K

Techniques theoretical basis1

Techniques/Theoretical Basis

Removal of other Cellular Macromolecules

  • CTAB-cetyltrimethylammonium bromide

  • NaCl

  • Chloroform/isoamyl Extraction

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

  • Quantitate amount of DNA isolated

  • Determine the Purity

  • Assess the Integrity

  • Set Up Restriction Digest

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