Amplification of Individual Phage Clones
This section describes how to amplify individual phage clones from infected E. coli colonies or liquid cultures. The amplification of individual phage clones is typically done between selection rounds in a biopanning (affinity selection) protocol to produce virions for further analysis. This method is also used to create fresh clones after prolonged storage.
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 amplification 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 when taking into account their optimal medium and antibiotic resistance.
- Terrific broth
- E. coli (K91BluKan)
- Kanamycin
- Tetracycline
- NZY medium
- NZY agar
Protocol
1. Inoculate 1 mL terrific broth with 50 µL an overnight culture of K91BluKan E. coli grown in NZY medium with 100 µg/mL kanamycin. Incubate the culture at 37º C and 250 rpm shaking until the OD600 of a 1/10 dilution reaches ~0.2 (late log phase). This will take approximately 2 h.
2. Slow the shaking down to 50 rpm for 5 min to allow sheared F pili to regenerate.
3. Add 1-100 µL of the phage solution to be amplified and continue slow shaking at 50 rpm for 15 min.
i. To create infected E. coli colonies from stock solutions of individual phage clones only a small volume (~1 µL) is needed.
ii. To create infected E. coli colonies from the eluates of biopanning experiments, or other solutions containing a diverse population of phage, a larger volume (~100 µL) is needed to prevent the loss of diversity.
4. Add 1 mL of NZY medium with 100 µg/mL kanamycin and incubate at 37ºC shaking at 200-250 rpm overnight.
5. Spread 20-200 µL of the overnight culture onto NZY agar plates with 100 µg/mL kanamycin and 40 µg/mL tetracycline. Let the plates dry and incubate overnight at 37ºC. E. coli colonies infected with phage should be visible after 18-24 h.
6. The plates can be stored at 4ºC for 1-3 weeks when sealed with parafilm.
Note: -Prepare pure phage virions: Small-Scale Phage Purification
Tips & Tricks
- When amplifying numerous individual phage clones, these can be plated on the same dish in a grid pattern using 20 µL of each culture. Special care should be taken to prevent cross-contamination.