Fingerprinting aquatic virus communities using denaturing gradient gel electrophoresis (DGGE)
Using lint-free tissues, wash glass plates, spacers, and combs thoroughly with 70% ethanol.
Assemble the gel sandwich by placing the small glass plate on top of the large plate, being sure to correctly place a 1-mm spacer along each edge of the plate assembly.
To prevent current leakage and the resultant “smiles” in the bands near the edges of the gel, grease both sides of the spacers with as little as possible silicon grease to cover the full length of the spacer but only a quarter of the spacer width.
Attach the plate clamps and place the entire assembly into the casting stand.
Inspect the plate assembly to ensure that the two glass plates and the spacers form a flush surface along the sides, and ensure that all gaskets adequately seal the plate assembly.
Check the gradient maker and flush with sdH2O.
Empty pump tubing and attach pipette tip at the outlet tube to the top-middle of the gel chamber.
To optimize the gradient conditions for a new DGGE experiment (new primer sets, new sample type/habitat, etc.), we usually start with a relatively broad gradient (20% to 80% denaturant).
We then focus the gradient around the area of interest to include the highest and lowest bands in different samples.
Table 1 (guidelines) can be used to determine the appropriate composition of the denaturing gradient gel (16 × 16 cm) that has a total volume of 29 mL.
Make up two solutions of 14.5 mL each, a low denaturant concentration solution and a high denaturant concentration solution.
Mix the solutions A and B (guidelines) to the desired percentage.
After preparing the denaturing gel solutions, degas for 15 min and filter through a 0.45-mm syringe filter.
Immediately before casting the gel, add 145 µL 10% APS and 7.25 µL TEMED into each solution and swirl gently to mix.
At this point, you will have approximately 10 min to pour the gel.
Make sure the pump is off and the gradient maker-channel is closed (handle up).
Pour solution with the highest denaturant in the right leg of the gradient maker (at the pump side) and the solution with the lowest denaturant in the left leg.
Turn the magnetic stirrer on, while simultaneously starting the pump (5 mL/min).
Simultaneously, start the pump (5 mL/min) and move the handle of the gradient maker to horizontal position (channel open).
Use approximately 4–5 min to fill the gel.
Empty the tubing and flush thoroughly with sdH2O.
Insert the comb, flat or straight side down, making sure that there are no bubbles under the comb.
Different combs (16 or 20 wells) are available, depending on the number of samples that you want to run.
Cover gels with cling film and allow ~2 h for the gel to polymerize.
The gel can be kept at 4°C until the next day.
Prepare approximately 7 L of 1× TAE and fill the buffer chamber.
Put about 0.5 L aside for later use.
To enhance the circulation of the running buffer, place the tank on a magnet stirrer and add a magnetic stirrer bar in the bottom of the tank.
Preheat the buffer in the DCode apparatus to 60°C; this will take about 2 h. Attach the gel plates to the core assembly.
Loosen the clamps a quarter-turn counterclockwise to prevent breaking of the sandwich clamps (due to heat expansion).
Then place the core assembly into the heated buffer in the tank.
Switch off the magnetic stirrer (if loading in tank).
Flush each well with buffer using syringe with needle to remove any unpolymerized acrylamide and excess urea.
Flush each well with buffer again before loading approximately 10–50 µL of PCR products mixed with loading dye into each well.
To quantify PCR products, we recommend gel quantification using a DNA mass standard (e.g., Low DNA Mass ladder; Invitrogen) and commercially available gel quantification software such as Quantity One (Bio-Rad) or free software such as Image J (available for download at http://rsbweb.nih.gov/ij/download.html).
In the DGGE gel, load a marker on each side of the gel adjacent to the samples (markers can be custom made for each DGGE application using known PCR products, or common molecular weight markers can be used)  for determination of band positions, or comparisons of different gels.
Apply a loading voltage of 200 V for 5 min before starting the pump to circulate the buffer, then turn on the magnetic stirrer.
The length of the run and the running voltage depend on the size of the PCR products and the percentage of acrylamide/bis in the gel.
A good starting point is to run the gel at 60 V (about 20 mA for one gel) for 19 h. 
When the electrophoresis is complete, take apart the apparatus and remove the glass plates from the gel clamps.
Carefully separate the plates, leaving the gel exposed on the large plate.
Use the edge of the small plate to trim the well walls, but be sure to leave the leftmost wall slightly higher than the others for use as a gel orientation reference.
For easy manipulation, the gel can either be stained on the large plate or transferred to, stained on, and transported on a plastic sheet.
Stain the gel for 30 min in 50–500 mL fluorescent gel stain.
Destain the gel for 30 min in 1 × TAE.
Remember, the fluorescent dye binds to nucleic acids; therefore, it is important to minimize contact with skin, so gloves (powder-free) should be worn.
If staining in a container, use plastic and not glass, as the fluorescent dyes accumulate over time on glass surfaces.
Slide the gel off of the plastic sheet or large plate onto a UV transilluminator and view the gel.
