Protocol for Pulsed Field Gel Electrophoresis
A 15-20 liter natural seawater sample is passed through a glass fiber pre-filter (Gelman A/E) and a 0.22 μm pore size membrane (Durapore, Millipore).
Concentrate to ca. 150-200 ml using 30 kD MWC Spiral Cartridge Concentrator (SCC).
Transfer the SCC retentate to Centriprep 30’s.
Store balance of concentrate in refrigerator.
Centrifuge in IEC-HN SE II at full speed for 15 minutes (in refrigerator if possible).
Empty central reservoir without refilling and repeat centrifugation step.
Empty and refill keeping volumes equal in order to maximize filtration area of Centripreps and balance rotor.
Continue to spin and empty until no more water enters central chamber.
Remove central insert from 2 of units, replace caps, and spin retentate chamber briefly to recover droplets from sides.
Combine retentate into 2, and finally one Centriprep unit, by gentle pipetting.
When all have been combined and spun, the final volume is ca. 500 μl.
Make a 1% agarose gel by combining 1 g Bio-Rad Molecular Grade Agarose with 99ml 0.5x TBE, and heating until completely clear of unmelted material.
Assemble mold with backing plate in place and pour in liquid.
Let set.
Meanwhile, fill electrophoresis chamber with 2 liters 0.5x TBE.
Turn on pump and chiller, and set temperature to 14°C.
When gel is set, remove comb and sides of mold, and slide backing plate, with gel attached, out of mold.
Place gel with backing plate into receiver in electrophoresis chamber, taking care not to dislodge gel from plate, with wells in rear and let chill.
Transfer aliquots into Microcons (30 or 100 kD MWC).
Place each aliquot in a separate Microcon and spin at 1000 x g for 20 minutes (about 4000 RPM in Eppendorf Microfuge) to near dryness (most of the Microcon membrane is dry, and only a small ring of liquid remains around edge).
As they reach the proper volume, smaller volume aliquots can be removed and stored in the refrigerator until the larger have been reduced to the proper volume.
When all samples have been reduced, add 50 μl 1:10 TE to each tube, taking care not to touch membrane with pipette tip, and spin again to reduce volume as described above.
Repeat step 22.
Repeat step 22 again.
Now add 20 μl 0.5x TBE to membrane to elute viruses, again taking care not to touch membrane.
Invert cartridge, place in fresh tube, and spin for 5 minutes at 1000 x g to recover.
Place tubes containing recovered viruses in 60°C water bath for 10 minutes.
Remove tubes and place immediately on ice for 2 minutes.
Transfer to microfuge and spin briefly to recover condensation from walls of tubes.
Prepare molecular weight markers by combining 100-200 ng of marker stock to 0.5x TBE to a final volume of 20 μl.
Add 10 μl PFGE loading buffer to each tube.
Mix by simultaneously inverting several times slowly, while rolling between index finger and thumb.
Turn chiller and pump off.
Load samples.
Turn pump on, then turn chiller on.
Close lid and check connections to make sure all is in order.
Set voltage to 6V.
Set initial switch time to 1s.
Set final switch time to 10s.
Set run time to 18h.
Push start.
After 10 minutes, check to make sure actual temperature is holding between 14 and 16°C, and that timer is running down.
