Restriction Enzyme Digest

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Restriction Enzyme Digest

• Digesting E. huxleyi genomic DNA

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Metacaspase

• Single or Multicopy gene in Emiliania huxleyi?

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

• Determine the concentration, purity, and
  integrity of the E. huxleyi genomic DNA
• Digest E. huxleyi genomic DNA

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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
• Aebc
• Where b light path length
• cconcentration of substance
• eextinction coefficient
• For DNA the extinction coefficient, e 50 ug/ml

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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

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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

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Pouring an Agarose Gel
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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)

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Assessing the Integrity of DNA
High Quality Genomic DNA gt95 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
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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)

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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

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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

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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

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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

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