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 : .
