Home Overview People Projects Protocols Sample collection for planktonic DNA DNA extraction from sediments Standard DGGE procedures Leucine incorporation rate, field procedures Methane production and oxidation rates Data/Posters/Reports Photos & Maps Publications Links	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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