1. Restriction Enzyme Digest
Digesting E. huxleyi genomic DNA

2. Single or Multicopy gene in Emiliania huxleyi?
Metacaspase
Single or Multicopy gene in Emiliania huxleyi?

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. BRAVO!!!
YOU DID IT!!!
You Isolated Genomic
DNA from E. huxleyi!!!

5. Today’s Laboratory Objectives
Determine the concentration, purity, and integrity of the E. huxleyi genomic DNA
Digest E. huxleyi genomic DNA

6. Theoretical Basis of UV Spectrophotometry
A UV spectophotometer measures the amount of light particular molecules absorb (Proteins at A280; Nucleic Acids at A260)
Lambert-Beer law describes the relationship between absorptivity coefficient and concentration and is given by the following equation:
A=εbc
Where: b= light path length
c=concentration of substance
ε=extinction coefficient
For DNA the extinction coefficient, ε= 50 ug/ml

7. Theoretical Basis of UV Spectrophotometry
To Quantify your DNA sample:
A260 x Dilution Factor x 50 ug/ml= concentration of nucleic acids in a sample using a 1 cm pathlength
To estimate the purity of your sample:
A260/A280= ratio of nucleic acids/protein
A260/A280= is optimal for DNA

8. 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

9. Pouring an Agarose Gel 3 1 2 4 5 6 7 8 9

10. Sizing a Piece of DNA The distance the DNA migrates is dependent upon
the size of the DNA molecule
the secondary structure of the DNA
the degree of crosslinking in the gel matrix
Size of DNA molecule can be determined by using standards of known molecular weight
a standard curve is made by plotting the molecular weights of the
standards and the distance each fragment has migrated from the measuring the distance the unknown fragment migrated from the
well
substituting the distance the unknown migrated into the equation of
the line of best fit, and solving for Y (the molecular wt)

11. 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

12. Restriction Enzymes
called "restriction enzymes“ because restrict host range for certain
bacteriophage
bacterial" immune system": destroy any "non-self" DNA
methylase recognizes same sequence in host DNA and protects it by methylating it; restriction enzyme destroys unprotected = non-self DNA (restriction/modification systems)

13. Restriction Enzymes
Hundreds of restriction enzymes have been identified.
Most recognize and cut palindromic sequences
Many leave staggered (sticky) ends
by choosing correct enzymes can cut DNA very precisely
Important for molecular biologists because restriction enzymes create unpaired "sticky ends" which anneal with any complementary sequence

14. Some Commonly Used Restriction Enzymes
Eco RI 5'-G | AATTC
Eco RV 5'-GAT | ATC
Hin D III 5'-A | AGCTT
Sac I 5'-GAGCT | C
Sma I 5'-CCC | GGG
Xma I 5'-C | CCGGG
Bam HI I 5'-G | GATCC
Pst I I 5'-CTGCA | G

15. Theoretical Basis Using Restriction Enzymes
The activity of restriction enzymes is dependent upon precise environmental condtions: PH
Temperature
Salt Concentration
Ions
An Enzymatic Unit (u) is defined as the amount of enzyme required to digest 1 ug of DNA under optimal conditions:
3-5 u/ug of genomic DNA
1 u/ug of plasmid DNA
Stocks typically at 10 u/ul

16. Next Week
Separate our restriction fragments using agarose gel electrophoresis
Southern Transfer- transfer denatured DNA from agarose gel to a membrane on which it can be analyzed using a labelled complementary DNA probe to metacaspase