1. Metacaspase Single or Multicopy gene in Emiliania huxleyi?

2. Isolation of Genomic DNA from Emiliania huxleyi CTAB Isolation and CsCl Gradient

3. 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

4. Today’s Objective To Isolate High Quality Genomic DNA from Emiliania huxleyi

5. Emiliania Huxleyi Unicellular eukaryotic marine algae Unicellular eukaryotic marine algae Calcium carbonate coccosphere Calcium carbonate coccosphere Blooms of > 100,000 sq kilometers Blooms of > 100,000 sq kilometers http://www.soes.soton.ac.uk/staff/tt/

6. What do we need to do to isolate genomic DNA?

7. Techniques/Theoretical Basis Breaking open E. huxleyi cells  SDS  Proteinase K

8. Techniques/Theoretical Basis Removal of other Cellular Macromolecules  CTAB-cetyltrimethylammonium bromide  NaCl  Chloroform/isoamyl Extraction

9. Techniques/Theoretical Basis  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 Cesium Chloride Gradient

10. 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 A 260 /A 280  Pure DNA A 260 /A 280 = 1.6-1.8

11. Next Week Quantitate amount of DNA isolated Quantitate amount of DNA isolated Determine the Purity Determine the Purity Assess the Integrity Assess the Integrity Set Up Restriction Digest Set Up Restriction Digest