Further Concentrating VCs for PFGE and other Techniques
DO NOT PUBLISH WITHOUT PERMISSION OF AUTHORS
K. Eric Wommack
A. Spin Filters (e.g., Centricon Plus 80, Centricon Plus 20)
1. Label one spin filter per sample to be concentrated. Be sure to label all parts of the spin filter (top, bottom, cap, recovery cap).
2. Pour each VC into the appropriately labeled spin filter. Fill it up, but leave enough room to put the cap on without overflowing.
3. Set up the table-top centrifuge (Eppendorf 5810 R) with the appropriate tube adapters (for Centricon 80s or Centricon 20s). Load filters.
4. Set run conditions: 4000 rpm (max for the centrifuge), 10 degrees C, acceleration rate 9, deceleration rate 9. Set time at 2 minutes for Centricon 20s, 5 minutes for Centricon 80s.
5. Start the run.
6. When run is complete, check the level of retentate. NEVER run the filters dry. Adjust the run time as needed to filter about 90% of the retentate volume. Drain the filtrate and refill the samples (retentate) as needed until the entire sample volume has been run through.
7. On the final run, (i.e., no more sample is waiting to be loaded into the spin filter) spin the tubes until no liquid can be seen freely floating above the filters. You should still be able to see liquid in the filter. A final wash can be performed with sterile buffer (e.g., SM buffer) if you prefer.
8. Insert the retentate recovery cap into the spin filter (see manufacturer’s instructions for more details). Invert the filter/recovery cap assembly and load into the centrifuge (inverted).
9. Set run conditions: 1000X g, time = 4 minutes, all other conditions as listed above. Start run.
10. When run is complete, there should be a small volume of viral concentrate in the bottom of the recovery cap. Use a 200ul (yellow) tip to carefully transfer to a sterile 2 ml eppendorf tube. Label the tube appropriately. Store at 4 degrees C until use.
B. Ultracentrifugation
1. Load SW 28 buckets with clean polyallomer tubes.
2. Pour VCs into tubes, keeping careful note of which VCs are in which tubes. Note also the weight of each tube and BALANCE opposite tubes to within +/- 1g.
3. Cap tubes and load into SW28 rotor. Be sure that tubes are firmly seated in the rotor.
4. Load rotor into centrifuge and run under the following conditions: 28 000 rpm, 4 degrees C, 12 hours, acceleration at max, deceleration at slow. Start run.
5. When run is complete and rotor has stopped, carefully remove tubes from rotor. Carefully pour or pipette off the supernatant, leaving about 5-10 ml of liquid in the bottom (a hazy film of phage may or may not be apparent on the tube bottoms). Pool the remaining 5-10 ml of phage concentrates in a 50 ml or 15 ml conical centrifuge tube. Make sure tubes are labeled.
6. Rinse each of the polyallomer tubes with 1 ml of SM, vortex briefly, and add to the pooled phage concentrate. Store at 4 degrees C until use.
