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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Definitions

• cDNA (complementary DNA)
• DNA copy of a gene that lacks introns and
  therefore consists solely of the coding sequence.
  Made by reverse transcription.
• cDNA Library
• Collection of genes in their cDNA form,
  lacking introns

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cDNA Library Construction
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Laboratory Objective

• To isolate plasmid containing a cDNA insert
  from Emiliania huxleyi, to use as a template in a
  DNA sequencing reaction

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Research Plan
Preparation of Competent Cells/Bacterial
Transformation
Growth of Transformant and Plasmid MiniPrep
DNA Sequencing
Sequence Analysis
BLASTN/BLASTX /ORF Finder/Clustal W
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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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Alkaline/SDS Cell Lysis

• SDS anionic detergent causes cell lysis
• Sodium hydroxide base, denatures DNA

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Selective Precipitation

• Potassium Acetate
• Ice
• Centrifugation

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Separation of Nucleic Acids

• RNase

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PEG Precipitation

• For Cleaner DNA Precipitate w/ Polyethylene
  Glycol and NaCl

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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 1/50
  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)