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Standard DGGE pocedures
Byron Crump, Nov. 15, 2001

Equipment:

  • PCR thermocycler
  • PCR tubes (sterile)
  • Horizontal agarose gel electrophoresis rig
  • Bio-Rad D-Code DGGE system with gel dimensions of 1 mm thick, 16 cm tall and 18 cm wide
  • Gradient maker with stir plate, stir bars, and peristaltic pump
  • 15 ml polypropylene conical centrifuge tubes

Solutions:

PCR solutions

  • RNAse-free sterile water
  • Taq Polymerase (Promega #M2665)
  • 10X PCR buffer B (comes with Promega Taq)
  • nucleotide mix of dTTP, dCTP, dATP, dGTP (10 mM each, BioRad #1708874)
  • Primers (10 uM)
    • 357f(g+c) (5'- CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-CCTACGGGAGGCAGCAG-3')
    • 519r (5'- ACCGCGGCTGCTGGCAC-3')
  • 2% agarose gel in 1X TAE running buffer (1x TAE is 40 mM Tris (pH 8.0), 20 mM acetic acid, 1 mM EDTA)

DGGE solutions

  • 8% acrylamide:bis-acrylamide with 0% denaturants
    • 54 ml 30% acrylamide:bis-acrylamide stock (37.5:1, Bio-Rad #1610158),
    • 2 ml 50x TAE buffer solution (Bio-Rad #1610743, final 0.5x)
    • bring to 200 ml with milli-Q water
  • 8% acrylamide:bis-acrylamide with 100% denaturants
    • 54 ml 30% acrylamide:bis-acrylamide stock (37.5:1, Bio-Rad #1610158),
    • 2 ml 50x TAE buffer solution (Bio-Rad #1610743, final 0.5x)
    • 80 ml 100% formamide (40% final concentration; Fisher super-pure BP228-100)
    • 84 g Urea (7M final concentration; electrophoresis grade, Fisher BP169)
  • TEMED (N,N,N',N'-Tetramethylethylenediamine; Sigma #T7024)
  • 10% w/v Ammonium persulfate (APS; BioRad #161-0700)
  • Bromphenol Blue in water (saturated solution)
  • Water-saturated butyl alcohol (Sigma#B1417)
  • Gel Loading Buffer
    • One recipe, for 10 ml:
    • 4 g sucrose (40% final)
    • 2 ml 0.5 M EDTA (100mM final)
    • 8 ml H2O
    • spatula each of bromophenol blue, xylene cyanol, and tartarazine
  • SYBR-gold stain (Molecular Probes #S-11494, 100 ul/1 L 0.5x TAE)

Procedure:

DGGE-PCR Amplification

  • Prepare PCR cocktail so each 50 ul reaction contains
    • 5 ul 10X PCR buffer
    • 5 ul dNTP mix
    • 5 ul 10 uM forward primer
    • 5 ul 10 uM reverse primer
    • 0.5 ul (2.5 U) Taq polymerase
    • emplate DNA (Amount optimized for each sample)
    • RNAse-free water to 50 ul
      • Thermocycle
    • 5 min at 94 degrees
    • 20-30 cycles of
      • 1 min at 94°C
      • 1 min at 65°C to 55°C (reduced by 0.5° per cycle for 20 cycles and further cycles at 55°C)
      • 1 min at 72°C
    • 5 min at 72°C.
  • NOTE: Steps involving a temperature reduction are done at 0.3°C/s. Optimize the number of cycles for each sample so that PCR is halted while product was still being produced exponentially (usually between 20 and 30 cycles).
  • Add 5 ul loading dye to PCR products
  • Quantify PCR products by running 3-5 ml on a 2% agarose gel with ladder

Gel Electrophoresis

  • Cast denaturing gradient acrylamide gels
    • Prepare acrylamide solutions of 30% and 50% denaturant in 15 ml tubes (11 ml for long combs, 12 ml for short combs)
    • Add 100 ul bromphenol blue solution to 30% solution.
    • Add 4.2 ul TEMED to each tube for 12 ml, 3.8 ul for 11 ml
    • Add 52 ul APS to each tube for 12 ml, 48 ul for 11 ml
    • Mix each quickly and pour into two towers of the gradient maker
    • Start stir bars in each tower
    • Begin pumping acrylamide from 50% tower
    • Open valve between towers when volume of 50% is visibly less than 30%
    • Top acrylamide between plates with 0.5-1 ml butyl alcohol while gel polymerizes 2+ hours
    • Gently rinse off butanol with distilled water and dry plates
    • Put in comb
    • Prepare stacking gel acrylamide
      • 5 ml 0% denaturant acrylamide (8 ml for 2- 11 ml gels)
      • 5 ul TEMED (8 ul for 8 ml)
      • 50 ul APS (80 ul for 8 ml)
    • Pipette stacking gel between plates and around comb
    • Allow stacking gel to polymerize (minimum 20 min.)
  • Load ladder in every 4th or 5th lane of DGGE gel.
  • Load similar quantities of PCR products in each lane.
  • Fill lanes (cover loaded samples) with 0.5x TAE buffer.
  • Fill DCode tank with 0.5x TAE buffer (buffer can be re-used 4-5x)
  • Electrophoresis is run at 65°C for 16-18 hours at 70 volts.

Visualization

  • Stain gels with SYBRgold for 1 hr in the dark
  • Photograph gels at a high resolution such that each band includes 5-10 pixels vertically. If this resolution cannot be achieved with one image, then take multiple images and reconstruct the gel image in Adobe Photoshop.


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