Small Scale Phage Purification
High-Throughput Phage Propagation and Isolation
This protocol is the routine procedure for propagating individual clones and is designed for processing up to 96 clones (1 mL cultures) simultaneously. The protocol can be scaled up and down (1-100 mL) to accommodate the propagation of different culture volumes.
This protocol is based on and modified from the The Laboratory of George P. Smith at the University of Missouri. The protocols were previously hosted by http://www.biosci.missouri.edu/smithgp/
Reagents
Note: This protocol describes the purification of filamentous phage based on the Fd-tet vector (tetracycline resistant) and uses K91BluKan E. coli cells (kanamycin resistant) that are cultured in NZY medium. The protocol can be modified to accommodate other phage variants and E. coli strains taking into account their optimal medium and antibiotic resistance.
- NZY medium
- Tetracycline
- Kanamycin
- PEG/NaCl
- TBS
Protocol
1. Add 1 mL NZY medium with 100 µg/mL kanamycin into the wells of a deep (2 mL) 96-well plate. Inoculate each well with E. coli infected with a single phage clone, either using an E. coli colony or 5 µL liquid E. coli culture. Incubate the plate overnight at 37ºC shaking at 200-250 rpm.
i. Important: The plate should be covered with a lid that allows air flow and prevents cross-contamination between the wells.
2. Centrifuge the 96-deep-well plate at 3000 xg for 2 min to pellet the cells.
i. Note: Alternatively, the cultures can be transferred to microcentrifuge tubes being careful not to cross-contaminate the samples.
3. Transfer the supernatant to a clean deep 96-well plate and add 0.15x volume PEG/NaCl solution. Mix thoroughly by pipetting and incubate at 4ºC for at least 4 h or ideally overnight.
4. Centrifuge at 8000 xg for 10 min to pellet the phage particles. Carefully, remove the supernatant by pipetting.
i. Note: The phage pellet will be small.
5. Dissolve the phage pellet in 1 mL TBS by vigorous pipetting. Centrifuge at 3000 xg for 2 min to pellet insoluble matter.
6. Transfer the supernatant to a fresh 96-deep-well plate and add 150 µL PEG/NaCl. Mix thoroughly by pipetting and incubate at 4ºC for at least 4 h or ideally overnight.
7. Centrifuge at 8000 xg for 10 min to pellet the phage particles. Carefully, remove the supernatant by pipetting. Note: Steps 5-7 may be repeated to achieve higher purity.
8. Dissolve the phage pellet in 10-100 µL TBS by pipetting. Transfer to a 96-well plate for storage at 4°C.
Notes: -Determine the physical particle concentration: Quantifying Phage Virions.
-Determine the number of transducing units:
Titering Transducing Units (TU).
Tips & Tricks
- Use a sticky plastic cover for the 96-well plate to prevent cross-contamination.
- Cross-contamination can easily occur and it is important to take all measurements to prevent this, including careful pipetting, opening of microcentrifuge lids and plate covers.
- Steps 5-7 can be repeated to provide a more pure phage solution.