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 .
