bot7

• DNA gels


  Used to separate fragments of DNA and RNA
• Agarose gel electrophoresis


  Submerged horizontal orientation, unlike most protein separations which use acrylamide polymers

  
• Principle of separation for all electrophoresis


  Movement of a charged molecule in a medium subjected to an electric field
• Velocity (v)


  Depends on electrical field (E), net charge on the molecule (q), and frictional coefficient (f)
• Frictional coefficient (f)


  Depends on the mass and shape of the molecule
• Agarose


  A seaweed extract (red algae agar) that forms a linear polymer with helical fibers and aggregates creating channels for DNA and RNA molecules to migrate through by 'reptation' (snaking)
• Mobility (μ)


  Relates to agarose concentration (i) according to the equation: log μ = log μ0 - Kri
• Agarose concentration and efficient range of separation of linear DNA molecules

  • 0.3% - 5 to 60 kb
  • 0.5% - 1 to 20 kb
  • 0.7% - 0.8 to 10 kb
  • 0.9% - 0.5 to 7 kb
  • 1.2% - 0.4 to 6 kb
  • 1.5% - 0.2 to 3 kb
  • 2.0% - 0.1 to 2 kb
• Supercoiled DNA


  Tightly wound double-stranded plasmid DNA that migrates faster than relaxed circular or linear DNA
• Relaxed circular DNA


  Plasmid DNA with one strand cut, migrates slower than supercoiled
• Linear DNA


  Plasmid DNA with both strands cut, migrates at its true molecular mass
• Single-stranded circular DNA


  Denatured plasmid DNA, migrates faster than supercoiled
• RNA contamination


  Runs as a wide band at a much smaller molecular mass than plasmid or genomic DNA
• Types of agarose


  • Molecular biology agarose
  • Low-melt agarose
  • PCR agarose
  • PCR low-melt agarose
• Molecular biology agarose


  General-purpose agarose with high exclusion limit, high gel strength, and ideal for preparative gels and DNA recovery
• Low-melt agarose


  Ideal for in-gel applications like ligation, PCR, restriction enzyme digestion, transformation, and sequencing
• PCR agarose


  High-strength agarose that forms flexible gels, ideal for DNA fragments <1,000 bp

  
• PCR low-melt agarose


  High sieving capacity, ideal for preparative electrophoresis and in-gel applications
• Buffers used for DNA gel electrophoresis

  • TAE
  • TBE
  • TPE
  • Na Borate

  
• TAE buffer


  Use when DNA is to be recovered, good for large >12 kb DNA, low ionic strength and buffering capacity
• TBE buffer


  Use for <1 kb DNA, provides tighter bands with higher % gels, high ionic strength and buffering capacity, not best for recovering DNA
• TPE buffer


  High buffering capacity, good for recovering DNA, good for long runs
• Na Borate buffer


  Used for high voltages providing faster runs, limited resolution, best for quick analytical gels
• EDTA is a chelator of divalent cations like Fe2+, Ca2+, and Mg2+ that are important for DNA enzyme activity and to limit metal-induced oxidation
• TAE buffer


  Best for quick analytical gels of purified DNA or restriction digests
• 50X TAE Stock preparation

  1. 242.0 g Tris Base

  2. 57.1 ml Glacial Acetic Acid

  3. 18.61g Na2EDTA.2H2O

  4. QS to 1.0 liter with water - do not adjust pH, but check...
• 1X TAE buffer


  40 mM Tris pH 7.6-8.0, 20 mM acetic acid, 1 mM EDTA
• 10X TBE Stock preparation

  1. 108.0 g Tris Base

  2. 55.0g boric acid

  3. 40 ml 0.5M EDTA (pH 8.0)

  4. QS to 1.0 liter with water
• 1X TBE buffer


  89 mM Tris pH 8.3, 89 mM boric acid, 2 mM EDTA
• 10X TPE Stock preparation

  1. 108.0 g Tris Base

  2. 15.5 ml 85% Phosphoric acid

  3. 7.44 g Na2EDTA.2H2O

  4. QS to 1.0 liter with water
• 1X TPE buffer


  89 mM Tris pH 8.3, 89 mM boric acid, 2 mM EDTA
• 1X Na Borate (SB) preparation

  1. Prepare 1M boric acid (6.1 g/100 ml water)

  2. Carefully add 1.0 ml of 10 M NaOH to 500 ml water with stirring

  3. Adjust pH of NaOH solution to pH 8.5 using a 1M Boric Acid slurry in a dropwise fashion
• Boric acid may not go into solution easily, slightly warm and/or use as a well mixed slurry
• Buffer Depth and Depletion: For any buffer the depth of buffer over the gel should range from 3 to 5 mm. Too much buffer will distort bands and cause heating and partial melting of the gel. Too little buffer and the gel is likely to partially dry out. Gel melting and band smearing is a tell-tale sign that the pH capacity of the buffer/gel has been depleted. This is mostly observed in longer runs in larger gels. Most mini-gels will not have this problem.
• Edge Effects / Smiling Gels: An uneven gel will cause issues with the electrical current subjected to the gel. Increased thickness of gel at the edge decreases resistance. Higher current causes more rapid migration of DNA at edges. Both of these will cause a smiling or sad (frowning) shape to the DNA gel. Often the outer gel lanes are avoided, as this effect hard to prevent.
• Gel Loading Dyes


  The dyes xylene cyanol FF (XC) and bromophenol blue (BB) plus 30% glycerol in water are often used to help visualize where your samples are during loading the gel and to find the "leading edge" and middle sized samples of your samples during electrophoresis. The glycerol makes the final solutions dense so they sink to the bottom of the wells.

  
• Using both dyes is helpful to see separation, BB is purpled and migrates at about the same rate as a linear double-stranded 400 bp DNA fragment whereas XC is blue-green and migrates at about the same rate as a 8000 bp DNA fragment. When BB reaches the end of the gel (about 2/3 of the way down the gel) the electrophoresis run is finished.

  
• 10X DNA loading dye preparation

  1. 0.025g Xylene cyanol FF (0.25% w/v)

  2. 0.025 g Bromophenol Blue (0.25% w/v)

  3. 5.0 ml glycerol (5%)

  4. 2 ml 500 mM EDTA (10 mM)

  5. QS to 10 ml with TAE buffer
• Substitutes for glycerol in DNA loading dye

  Sucrose (40%), Ficol (25-30%) or glycerol (30-50%)
• Optional additions to DNA loading dye

  • 1 ml of 10% SDS to eliminate protein-DNA interactions
  • 100 mM (in 10X stock) EDTA to avoid enzymatic degradation of DNA/RNA