Plasmid Miniprep

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Plasmid Miniprep
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Broad and Long Term Objective

• To characterize a single clone from an
• Emiliania huxleyi cDNA library using
• sequence analysis

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Research Plan
Preparation of Competent Cells/Bacterial
Transformation
Growth of Transformant and Plasmid MiniPrep
DNA Sequencing
Sequence Analysis
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Todays Laboratory Objectives

• To isolate high quality plasmid DNA that can be
  used as
• template for DNA sequencing
• To quantify and determine the purity of the
  isolated
• plasmid DNA
• To determine the size of the plasmid DNA and its
  insert

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Map of Parent Vector pMAB58
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Theoretical Basis of the Alkaline Lysis Plasmid
Miniprep

• 1. Lyse Cells
• 2. Separate nucleic acids from other cellular
  macromolecules
• 3. Concentrate nucleic acids
• 4. Separate RNA from DNA

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1. Lyse Cells

• Alkaline/SDS Cell Lysis
• SDS anionic detergent that solubulizes
• membranes and denatures proteins
• Sodium hydroxide pH to 12.0,
• denaturation of DNA

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2. Separation of nucleic acids from other
cellular macromolecules
NaOH
K-acetate
protein
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3. Concentration of Nucleic Acids
Precipitation 1 Isopropanol cations (Na)
Precipitation 2 PEG cations (Na) (increased
purity for sequencing)
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4. Separation of RNA from DNA
RNaseA
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Theoretical Basis of UV Spectrophotometry

• A UV spectophotometer measures the amount of
  light particular molecules absorb (Proteins at
  280 nm Nucleic acids at 260 nm)
• Lambert-Beer law describes the relationship
  between absorbtivity coefficient and
  concentration and is given by the following
  equation
• Aebc
• Where b light path length (cm)
• cconcentration of substance
• (ug/ml)
• eextinction coefficient
• For DNA the extinction
• coefficient, e _____1_____
• 50 ug/ml cm

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Theoretical Basis of UV Spectrophotometry

• To Quantify your DNA sample
• Abs 260nm x Dilution Factor x 50 ug/ml
  concentration of DNA 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 restriction endonuclease
digestion

• Type II restriction endonucleases cleave
• DNA at specific, usually palindromic,
• sequences

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Theoretical Basis of Agarose Gel Electrophoresis

• Agarose is a polysaccharide from marine algae
  that forms a matrix allowing separation of DNA
  molecules
• Because DNA is a (-) charged molecule, when
  subjected to an electric current it will migrate
  towards a () pole
• Separation based upon DNA size and secondary
  structure

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Pouring an Agarose Gel
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Sizing a Piece of DNA

• Size of DNA molecule can be determined by using
  standards of known size
• 1. A standard curve is made by plotting the
  size (in
• bp) of the standards (Y-axis) against the
  distance
• each fragment has migrated from the well
  (X-
• axis)
• 2. Measure 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 size)