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 .
