Fingerprinting aquatic virus communities using pulsed field gel electrophoresis (PFGE)
Prepare the 1.5% agarose and the lysis buffer.
Incubate the agarose at 80°C until further use.
Dispense 5 mL of the freshly made lysis buffer into 50-mL Falcon tubes, one for each sample.
Combine equal volumes (200 µL) of virus concentrate and molten 1.5% agarose.
Mix briefly.
Dispense the mixture into plug molds with a pipette.
Place the plug molds in the freezer (–20°C) for at least 2 min to set.
Remove the tape from the bottom of the plug molds and push the plugs out from the molds into 5 mL lysis buffer.
Incubate the plugs in lysis buffer overnight in the dark at 30°C.
The next day, decant the lysis buffer using a plastic sieve (screened cap) that can be attached at the top of the Falcon tube.
Wash the plugs three times, 30 min each, in TE buffer 10:1 at room temperature.
The plugs can be stored at 4°C in TE 20:50 for several month before further processing; nevertheless, we recommend running the samples as soon as possible, because degradation of the viral DNA will occur and result in less discrete bands.
Set up the gel rig.
Prepare a 1% agarose gel in 1× TBE buffer.
Melt until the agarose is completely dissolved.
Place the warm agarose at 60°C for 10 min before pouring into the gel rig and allowing it to cool.
Keep ~5 mL agarose at 60°C for later use to seal the wells.
When the agarose is set, pour 50 mL of 1× TBE running buffer on the top of the gel and place it in the refrigerator for at least 20 min or overnight.
Place molecular weight standards (slices of ~5 mm) on either side of the gel.
Place the samples between the markers using a sterile loop.
Overlay the wells with leftover molten 1% agarose.
Remove the gel from the pouring rig and remove any extraneous agarose from the bottom and edges.
Prepare the 1× TBE (running buffer) and place at 14°C until further use.
Place the gel into the electrophoresis chamber and carefully pour the cooled 1× TBE running buffer into the chamber.
Run the gel at 6 V cm–1 with pulse ramps from 20 to 40 s (for example) at 14°C for 22 h. To separate different viral genome size classes, each sample could be run three times:.
