Kiffy

• Electrophoresis is the movement of molecules by an electric current
• Under an electric current, DNA and RNA will migrate toward the positive pole (anode)
• Double-stranded DNA and RNA


  Analyzed by native gel electrophoresis
• Single-stranded DNA and RNA

  Form hairpin-like structures and heteroduplexes when complementary strands are not available
• Ame Tiselius developed an electrophoresis apparatus while studying resolution diffusion and adsorption of proteins in naturally occurring porous silicate particles
• Oliver Smithies found that if he heated about 15 grams of starch in 100 mL of his electrophoretic medium and allowed it to cool, he could make a gel matrix with resolving properties similar to a paper electrophoresis method he had previously devised
• In 1956, Smithies and Poulik described the resolution of 20 serum protein components using a two-dimensional electrophoresis system with paper in one dimension and starch in the other
• Agarose


  A polysaccharide polymer extracted from seaweed, a component of agar used in bacterial culture dishes
• Agarose gels

  • Small pieces of DNA (50 to 500 base pairs) are resolved on more concentrated agarose gels (2% to 3%)
  • Larger fragments of DNA (2,000 to 50,000) are best resolved in lower agarose concentrations (0.5% to 1%)
  • Agarose concentrations above 5% and below 0.5% are not practical
• Agarose preparations are sufficiently pure to avoid problems such as electroendosmosis, a solvent flow toward one of the electrodes usually the cathode (negative), in opposition to the DNA or RNA migration
• Pulsed-Field Gel Electrophoresis can resolve very large pieces (50,000 to 250,000 + bp) of DNA that cannot be resolved efficiently by simple agarose electrophoresis
• Polyacrylamide gels


  Used to resolve very small DNA fragments, single-stranded DNA, RNA, and proteins
• Polyacrylamide gels


  • Unlike agarose, polyacrylamide is a synthetic material allowing precise control of the polymer properties and higher resolution
  • Polyacrylamide gels require a catalyst like ammonium persulfate (APS) plus N,N,N',N' tetramethylethylenediamine (TEMED) or light activation to polymerize
• Capillary electrophoresis


  • Separation based on size and charge (charge/mass ratio)
  • Faster analytical runs and lower cost per run than other separation methods
  • Fluorescent labels are covalently attached to nucleic acids for detection
• Buffer


  A solution of a weak acid and its conjugate base that carries the current and protects the samples during electrophoresis
• Henderson-Hasselbalch equation


  Used to calculate the pH of a solution containing a weak acid and its conjugate base, or a weak base and its conjugate acid
• Sample Problem #1

  Calculate the pH of a buffer solution containing 0.1 M acetic acid (CH3COOH) and 0.05 M sodium acetate (CH3COONa), given the pKa of acetic acid is 4.76
• Sample Problem #2


  Calculate the dissociation constant (Ka) of acetic acid (CH3COOH) in a buffer solution containing 0.1 M acetic acid (CH3COOH) and 0.05 M sodium acetate (CH3COONa), given the pH of the buffer solution is 4.74
• Sample Problem #3

  Calculate the pKa of acetic acid (CH3COOH) given the dissociation constant (Ka) is 1.8 x 10^-5
• Buffer systems

  • Buffer protects sample molecules from damage and carries the current through the gel
  • Buffer concentration must be high enough to provide sufficient acidic and basic forms to buffer its solution, but not too high to generate excessive heat
  • Tris base or borate are preferred buffer components as they remain partly uncharged at the desired pH
• TBE buffer


  Has a greater buffering capacity than TAE, but stock solutions are prone to precipitation
• TPE buffer


  Can overheat when run at high voltage in a closed container
• TAE buffer


  More easily exhausted during extended or high-voltage electrophoresis, but DNA migrates twice as fast in TAE than in TBE in a constant current
• Formamide and urea


  Added to DNA and RNA to break and block hydrogen-bonding sites, hindering complementary sequences from reannealing
• Fluorescent dyes


  Intercalating agents like ethidium bromide, minor groove-binding dyes like SYBR Green, and silver stain for protein visualization