Restriction Enzyme Digest Digesting E. huxleyi genomic DNA
Metacaspase Single or Multicopy gene in Emiliania huxleyi?
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
BRAVO!!! YOU DID IT!!! You Isolated Genomic DNA from E. huxleyi!!!
Today’s Laboratory Objectives • Determine the concentration, purity, and integrity of the E. huxleyi genomic DNA • Digest E. huxleyi genomic DNA
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
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= 1.6-1.8 is optimal for DNA
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
Pouring an Agarose Gel 3 1 2 4 5 6 7 8 9
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 1. a standard curve is made by plotting the molecular weights of the standards and the distance each fragment has migrated from the 2. measuring the distance the unknown fragment migrated from the well 3. substituting the distance the unknown migrated into the equation of the line of best fit, and solving for Y (the molecular wt)
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
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)
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
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
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
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